U.S. patent number 7,635,472 [Application Number 10/554,852] was granted by the patent office on 2009-12-22 for pharmaceutical compositions comprising bispecific anti-cd3, anti-cd19 antibody constructs for the treatment of b-cell related disorders.
This patent grant is currently assigned to Micromet AG. Invention is credited to Patrick Bauerle, Birgit Kohleisen, Peter Kufer, Ralf Lutterbuse, Steven Zeman.
United States Patent |
7,635,472 |
Kufer , et al. |
December 22, 2009 |
**Please see images for:
( Certificate of Correction ) ** |
Pharmaceutical compositions comprising bispecific anti-cd3,
anti-cd19 antibody constructs for the treatment of b-cell related
disorders
Abstract
The present invention relates to a pharmaceutical composition
comprising a bispecific single chain antibody construct comprising
binding domains specific for human CD3 and human CD19, wherein the
corresponding variable heavy chain regions (V.sub.H) and the
corresponding variable light chain regions (V.sub.L) regions are
arranged, from N-terminus to C-terminus, in the order
V.sub.H(CD19)-V.sub.L(CD19)-V.sub.H(CD3)-V.sub.L(CD3) or
V.sub.H(CD3)-V.sub.L-(CD3)-V.sub.H(CD19)-V.sub.L (CD19). Processes
for the production of the pharmaceutical compositions and
medical/pharmaceutical uses for the specific bispecific single
chain antibody molecules bearing specificities for the human CD3
antigen and the human CD19 antigen are also disclosed.
Inventors: |
Kufer; Peter (Moosburg,
DE), Lutterbuse; Ralf (Munich, DE),
Kohleisen; Birgit (Munich, DE), Zeman; Steven
(Munich, DE), Bauerle; Patrick (Gauting,
DE) |
Assignee: |
Micromet AG (Munchen,
DE)
|
Family
ID: |
33483898 |
Appl.
No.: |
10/554,852 |
Filed: |
May 26, 2004 |
PCT
Filed: |
May 26, 2004 |
PCT No.: |
PCT/EP2004/005685 |
371(c)(1),(2),(4) Date: |
April 04, 2006 |
PCT
Pub. No.: |
WO2004/106381 |
PCT
Pub. Date: |
December 09, 2004 |
Prior Publication Data
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|
|
Document
Identifier |
Publication Date |
|
US 20070123479 A1 |
May 31, 2007 |
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Foreign Application Priority Data
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|
|
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May 31, 2003 [EP] |
|
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03012136 |
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Current U.S.
Class: |
424/130.1;
530/387.1 |
Current CPC
Class: |
A61P
31/04 (20180101); A61P 31/12 (20180101); A61P
37/08 (20180101); C07K 16/2803 (20130101); A61P
19/02 (20180101); A61P 31/00 (20180101); A61P
35/00 (20180101); A61P 35/02 (20180101); A61P
37/02 (20180101); A61P 37/06 (20180101); A61P
37/04 (20180101); A61P 33/00 (20180101); A61P
37/00 (20180101); A61P 29/00 (20180101); A61P
43/00 (20180101); C07K 2317/31 (20130101); A61K
2039/505 (20130101); C07K 2317/34 (20130101); C07K
2317/622 (20130101) |
Current International
Class: |
A61K
39/395 (20060101); C07K 16/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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195 31 348 |
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Feb 1997 |
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DE |
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0 505 908 |
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Sep 1992 |
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EP |
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1293514 |
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Mar 2003 |
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EP |
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WO 91/09968 |
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Jul 1991 |
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WO |
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WO 95/11922 |
|
May 1995 |
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WO |
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WO 96/36360 |
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Nov 1996 |
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WO |
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WO 99/54440 |
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Oct 1999 |
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WO |
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WO 02/16414 |
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Feb 2002 |
|
WO |
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|
Primary Examiner: Ouspenski; Ilia
Attorney, Agent or Firm: Foley & Lardner LLP
Claims
The invention claimed is:
1. A pharmaceutical composition comprising a bispecific single
chain antibody construct, said bispecific single chain antibody
construct comprising binding domains specific for human CD3 and
human CD19, wherein the corresponding variable heavy chain regions
(V.sub.H) and the corresponding variable light chain regions
(V.sub.L) regions are arranged, from N-terminus to C- terminus, in
the order, V.sub.H(CD19)-V.sub.L(CD19)-V.sub.H(CD3)-V.sub.L(CD3),
or V.sub.H(CD3)-V.sub.L(CD3)-V.sub.H(CD19)-V.sub.L(CD19).
2. The pharmaceutical composition of claim 1, wherein said V.sub.H
and V.sub.L regions of said CD3 specific domain are derived from an
CD3 specific antibody consisting of: OKT-3.
3. The pharmaceutical composition of claim 1, wherein said V.sub.H
region comprises at least one CDR3 region comprising the amino acid
sequence: SEQ ID NO: 54; at least one CDR2 region comprising the
amino acid sequence: SEQ ID NO: 53; and at least one CDR1 region
comprising the amino acid sequence: SEQ ID NO: 52; and wherein said
V.sub.L region comprises at least one CDR3 region comprising the
amino acid sequence: SEQ ID NO: 57; at least one CDR2 region
comprising the amino acid sequence: SEQ ID NO: 56; and at least one
CDR1 region comprising the amino acid sequence: SEQ ID NO: 55.
4. The pharmaceutical composition of claim 1, wherein said
bispecific single chain antibody construct comprises an amino acid
sequence selected from the group consisting of (a) an amino acid
sequence as depicted in SEQ ID NO: 2; (b) an amino acid sequence
encoded by a nucleic acid sequence as shown in SEQ ID NOs: 1, 9 or
13; and (c) an amino acid sequence encoded by a nucleic acid
sequence which is degenerate as a result of the genetic code to a
nucleotide sequence of (b).
5. The pharmaceutical composition of claim 1, wherein said variable
domains are connected by additional linker sequences.
6. A pharmaceutical composition claim 1 further comprising a
proteinaceous compound capable of providing an activation signal
for immune effector cells, wherein the proteinaceous compound is
selected from the group consisting of scFv fragments specific for
4-1BB, OX 40, CD27, CD70, the receptors for B7-RP1, B7-H3 and scFv
fragments specific for the T cell receptor or superantigens.
7. A process for the production of a pharmaceutical composition of
claim 1, said process comprising culturing a host cell under
conditions allowing the expression of a bispecific single chain
antibody construct comprising binding domains specific for human
CD3 and human CD 19, wherein the corresponding variable heavy chain
regions (V.sub.H) and the corresponding variable light chain
regions (V.sub.L) are arranged, from N-terminus to C- terminus, in
the order V.sub.H(CD19)-V.sub.L(CD19)-V.sub.H(CD3)-V.sub.L(CD3) or
V.sub.H(CD3)-V.sub.L(CD3)-V.sub.H(CD19)-V.sub.L(CD19), recovering
the produced bispecific single chain antibody construct from the
culture, and producing the pharmaceutical composition.
8. A kit comprising a bispecific single chain antibody construct as
defined in claim 1.
Description
This application is a National Stage application of
PCT/EP2004/005685 filed May 26, 2004, which claims priority from
European patent application EP 03012136.2, filed May 31, 2003. The
entire contents of each of the aforementioned applications are
incorporated herein by reference.
The present invention relates to a pharmaceutical composition
comprising a bispecific single chain antibody construct, said
bispecific single chain antibody construct comprising binding
domains specific for human CD3 and human CD19, wherein the
corresponding variable heavy chain regions (V.sub.H) and the
corresponding variable light chain regions (V.sub.L) are arranged,
from N-terminus to C-terminus, in the order,
V.sub.H(CD19)-V.sub.L(CD19)-V.sub.H(CD3)-V.sub.L(CD3),
V.sub.H(CD3)-V.sub.L(CD3)-V.sub.H(CD19)-V.sub.L(CD19) or
V.sub.H(CD3)-V.sub.L(CD3)-V.sub.L(CD19)-V.sub.H(CD19). Furthermore,
processes for the production of said pharmaceutical compositions as
well as medical/pharmaceutical uses for the specific bispecific
single chain antibody molecules bearing specificities for the human
CD3 antigen and the human CD19 antigen are disclosed.
Despite the medical importance, research in B-cell mediated
diseases such as non-Hodgkin lymphoma has produced only a small
number of clinically usable data and conventional approaches to
cure such diseases remain tedious and unpleasant and/or have a high
risk of relapse. For example, although high dose chemotherapy as a
primary treatment for high grade non-Hodgkin lymphoma may improve
overall survival, about 50% of the patients still die of this
disease (Gianni, N Engl. J. Med. 336 (1997), 1290-7; Urba, J. Natl.
Cancer Inst. Monogr. (1990), 29-37; Fisher, Cancer (1994)).
Moreover, low-grade non-Hodgkin lymphoma-like chronic lymphatic
leukemia and mantle cell lymphoma are still incurable. This has
stimulated the search for alternative strategies such as
immunotherapy. Antibodies directed against cell surface molecules
defined by CD antigens represent a unique opportunity for the
development of therapeutic reagents. The expression of certain CD
antigens is highly restricted to specific lineage
lymphohematopoietic cells and over the past several years,
antibodies directed against lymphoid-specific antigens have been
used to develop treatments that were effective either in vitro or
in vivo animal models (Bohlen, Blood 82 (1993), 1803-121; Bohlen,
Cancer Res 53 (1993), 18: 4310-4; Bohlen, Cancer Res 57 (1997),
1704-9; Haagen, Clin Exp Immunol 90 (1992), 368-75; Haagen, Cancer
Immunol Immunother. 39 (1994), 391-6; Haagen, Blood 84 (1994),
556-63; Haagen, Blood 85 (1995), 3208-12; Weiner, Leuk Lymphoma 16
(1995), 199-207; Csoka, Leukemia 10 (1996), 1765-72.). In this
respect CD19 has proved to be a very useful target. CD19 is
expressed in the whole B lineage from the pro B cell to the mature
B cell, it is not shed, is uniformly expressed on all lymphoma
cells, and is absent from stem cells (Haagen, Clin Exp Immunol 90
(1992), 368-75; Uckun, Proc. Natl. Acad. Sci. USA 85 (1988),
8603-7). An interesting modality is the application of a bispecific
antibody with one specificity for CD19 and the other for the CD3
antigen on T cells. However, bispecific antibodies thus far
available suffer from low T-cell cytotoxicity and the need of
costimulatory agents in order to display satisfactory biological
activity. The CD3 complex denotes an antigen that is expressed on
T-cells as part of the multimolecular T-cell receptor complex. It
consists of several different chains for instance .gamma., .delta.,
.epsilon., .zeta. or/and .eta. chains. Clustering of CD3 on T
cells, e.g., by immobilized anti-CD3-antibodies, leads to T cell
activation similar to the engagement of the T cell receptor but
independent from its clone typical specificity. Actually, most
anti-CD3-antibodies recognize the CD3.epsilon.-chain.
Prior art has exemplified T cell activation events employing
antibody molecules. For example, U.S. Pat. No. 4,361,549 proposes a
hybrid cell line for the production of monoclonal antibody to an
antigen found on normal human T cells and cutaneous T lymphoma
cells and defines the antibody produced as "OKT3". In U.S. Pat. No.
5,885,573 the murine OKT3 (described in U.S. Pat. No. 4,361,549)
has been transferred into a human antibody framework in order to
reduce its immunogenicity. Furthermore, U.S. Pat. No. 5,885,573
discloses specific mutations in the Fc receptor ("FcR")-binding
segment of OKT-3 which leads to a Glu at position 235, a Phe at
position 234 or a Leu at position 234, i.e. to specific mutations
in the CH2 region which are supposed to result in modified binding
affinities for human FcR. In proliferation assays or in assays
relating to the release of cytokines, the mutated OKT-3 antibodies
disclosed in U.S. Pat. No. 5,885,573 appear to result in comparable
cell proliferations to that observed with PBMC stimulated with the
original murine OKT3 and to similar amounts of cytokines produced.
Merely the mutated Glu-235 mAb induced smaller quantities of
TNF-.alpha. and GM-CSF and no IFN-.gamma.. No T cell proliferation
was induced by Glu-235 monoclonal antibody ("mab") using PBMC from
three different donors at mab concentrations up to 10 .mu.g/ml,
suggesting that the alteration of the FcR binding region of this
mab had impaired its mitogenic properties. T cell activation by
Glu-235 mab also resulted in lower levels of expression of surface
markers Leu23 and IL-2 receptor. U.S. Pat. No. 5,929,212 discloses
a recombinant antibody molecule in which the binding regions have
been derived from the heavy and/or light chain variable regions of
a murine anti-CD3 antibody, e.g. OKT3, and have been grafted into a
human framework. WO 98/52975 discloses a mutated variant of the
murine anti-CD3 antibody OKT3. The mutated OKT3 antibody is
produced using a recombinant expression system and WO 98/52975
proposes that the mutated anti-CD3 antibody is more stable than the
parental OKT3 protein during extended storage periods. U.S. Pat.
No. 5,955,358 discloses a method of shuffling, at the DNA level,
multiple complementarity determining ("CDR") domains, either from
the same or different antibodies, meaning that their order within
antibody variable domains is altered to yield new combinations of
binding regions.
OKT3 has been used as potent immunosuppressive agent in clinical
transplantation to treat allograft rejection (Thistlethwaite 1984,
Transplantation 38, 695-701; Woodle 1991, Transplantation 51,
1207-1212; Choi 2001, Eur. J. Immunol. 31(1), 94-106). Major
drawbacks of this therapy are T cell activation manifested in
cytokine release due to cross-linking between T cells and
Fc.gamma.R-bearing cells and the human anti-mouse antibody (HAMA)
response. Several publications have described alterations such as
humanization of OKT3 to reduce these side effects: U.S. Pat. No.
5,929,212; U.S. Pat. No. 5,885,573 and others. On the other hand,
OKT3 or other anti-CD3-antibodies can be used as immunopotentiating
agents to stimulate T cell activation and proliferation (U.S. Pat.
No. 6,406,696 Bluestone; U.S. Pat. No. 6,143,297 Bluestone; U.S.
Pat. No. 6,113,901 Bluestone; Yannelly 1990, J. Immunol. Meth. 1,
91-100). Anti-CD3-antibodies have also been described as agents
used in combination with anti-CD28-antibodies to induce T cell
proliferation (U.S. Pat. No. 6,352,694). OKT3 has further been used
by itself or as a component of a bispecific antibody to target
cytotoxic T cells to tumor cells or virus infected cells (Nitta
1990, Lancet 335, 368-376; Sanna 1995, Bio/Technology 13,
1221-1224; WO 99/54440).
Approaches up to now using antibodies as agents for recruiting
T-cells have been hampered by several findings. First, natural or
engineered antibodies having a high binding affinity to T-cells
often do not activate the T-cells to which they are bound. Second,
natural or engineered antibodies having a low binding affinity to
T-cells are also often ineffective with respect to their ability to
trigger T-cell mediated cell lysis.
Bispecific antibodies comprising specificities for human CD19 and
human CD3 which are not of the single-chain format and which
retarget T-cell cytotoxicity to lymphoma cells in an
MHC-independent manner have already been shown to be effective in
vivo in animal models (Bohlen, Cancer Res 57 (1997), 1704-9;
Demanet, Int J Cancer Suppl 7 (1992), 67-8) as well as in some
pilot clinical trials. So far these antibodies were constructed by
hybrid-hybridoma techniques, by covalently linking the monoclonal
antibodies (Anderson, Blood 80 (1992), 2826-34) or by a diabody
approach (Kipriyanov, Int. J. Cancer 77 (1998), 763-772). More
extensive clinical studies have been hampered by the fact that
these antibodies have low biological activity such that high
dosages have to be administered and that application of the
antibodies alone did not provide for a beneficial therapeutic
effect. Furthermore, the availability of clinical grade material
was limited. The prior art has exemplified bispecific single chain
antibodies comprising specificities for both human CD3 and human
CD19 antigens (Loffler, Blood 95 (2000), 2098-103; WO 99/54440;
Dreier, Int. J. Cancer. 100 (2002), 690-7). WO 99/54440 documents
the successful clinical use of a construct in the format
V.sub.L(CD19)-V.sub.H(CD19)-V.sub.H(CD3)-V.sub.L(CD3) and stresses
that the order of variable domains within the construct is not
decisive.
Yet, in particular for distinct clinical and pharmaceutical uses,
constructs have to be provided which can be produced in large
amounts by reasonably high levels of expression of the recombinant
constructs and by adequate purification methods after expression.
In the event that extremely low amounts of pure protein are
obtained, it becomes prohibitively cumbersome and/or costly to
generate therapeutically relevant amounts of such constructs. In
the special case of proteinaceous medicaments intended for parental
administration, these medicaments should be highly active and
potent, even in low concentrations, in order to avoid adverse
side-effects due to excessive protein concentrations or voluminous
infusion/injection solutions. Disadvantages of highly-dosed
proteinaceous medicaments or highly-dosed medicaments based on
nucleic acids comprise, inter alia, the promotion of
hypersensitivities and inflammatory events, in particular at the
site of administration. Thus, the technical problem of the present
invention is the provision of means and methods for the generation
of well tolerated and convenient medicaments for the treatment and
or amelioration of B-cell related or B-cell mediated disorders.
Accordingly, the present invention relates to a pharmaceutical
composition comprising a bispecific single chain antibody
construct, said bispecific single chain antibody construct
comprising binding domains specific for human CD3 and human CD19,
wherein the corresponding variable heavy chain regions (V.sub.H)
and the corresponding variable light chain regions (V.sub.L) are
arranged, from N-terminus to C-terminus, in the order,
V.sub.H(CD19)-V.sub.L(CD19)-V.sub.H(CD3)-V.sub.L(CD3),
V.sub.H(CD3)-V.sub.L(CD3)-V.sub.H(CD19)-V.sub.L(CD19) or
V.sub.H(CD3)-V.sub.L(CD3)-V.sub.L(CD19)-V.sub.H(CD 19).
Accordingly, "V.sub.L" and "V.sub.H" means the variable domain of
the light and heavy chain of specific anti-CD19 (CD19) and anti-CD3
(CD3) antibodies.
In accordance with this invention, the term "pharmaceutical
composition" relates to a composition for administration to a
patient, preferably a human patient. In a preferred embodiment, the
pharmaceutical composition comprises a composition for parenteral,
transdermal, intraluminal, intraarterial, intrathecal
administration or by direct injection into tissue. It is in
particular envisaged that said pharmaceutical composition is
administered to a patient via infusion or injection. Administration
of the suitable compositions may be effected by different ways,
e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular,
topical or intradermal administration. The pharmaceutical
composition of the present invention may further comprise a
pharmaceutically acceptable carrier. Examples of suitable
pharmaceutical carriers are well known in the art and include
phosphate buffered saline solutions, water, emulsions, such as
oil/water emulsions, various types of wetting agents, sterile
solutions, etc. Compositions comprising such carriers can be
formulated by well known conventional methods. These pharmaceutical
compositions can be administered to the subject at a suitable dose.
The dosage regiment will be determined by the attending physician
and clinical factors. As is well known in the medical arts, dosages
for any one patient depend upon many factors, including the
patient's size, body surface area, age, the particular compound to
be administered, sex, time and route of administration, general
health, and other drugs being administered concurrently. A
preferred dosage for administration might be in the range of 0.24
.mu.g to 48 mg, preferably 0.24 .mu.g to 24 mg, more preferably
0.24 .mu.g to 2.4 mg, even more preferably 0.24 .mu.g to 1.2 mg and
most preferably 0.24 .mu.g to 240 .mu.g units per kilogram of body
weight per day. Particularly preferred dosages are recited herein
below. Progress can be monitored by periodic assessment. Dosages
will vary but a preferred dosage for intravenous administration of
DNA is from approximately 10.sup.6 to 10.sup.12 copies of the
nucleic acid molecule, preferably a DNA molecule. The
pharmaceutical compositions of the invention comprising
proteinaceous or nucleic acid compounds described herein may be
administered locally or systematically. Administration will
generally be parenterally, e.g., intravenously; DNA may also be
administered directed to the target site, e.g., by biolistic
delivery to an internal or external target site or by catheter to a
site in an artery. Preparations for parenteral administration
include sterile aqueous or non-aqueous solutions, suspensions, and
emulsions. Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oils such as olive oil, and
injectable organic esters such as ethyl oleate. Aqueous carriers
include water, alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media. Parenteral
vehicles include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride, lactated Ringer's, or fixed oils.
Intravenous vehicles include fluid and nutrient replenishers,
electrolyte replenishers (such as those based on Ringer's
dextrose), and the like. Preservatives and other additives may also
be present such as, for example, antimicrobials, anti-oxidants,
chelating agents, inert gases and the like. In addition, the
pharmaceutical composition of the present invention might comprise
proteinaceous carriers, like, e.g., serum albumin or
immunoglobulin, preferably of human origin. It is envisaged that
the pharmaceutical composition of the invention might comprise, in
addition to the proteinaceous bispecific single chain antibody
constructs or nucleic acid molecules or vectors encoding the same
(as described in this invention), further biologically active
agents, depending on the intended use of the pharmaceutical
composition. Such agents might be drugs acting on the
gastrointestinal system, drugs acting as cytostatica, drugs
preventing hyperurikemia, drugs inhibiting immunoreactions (e.g.
corticosteroids), drugs acting on the circulatory system and/or
agents such as T-cell co-stimulatory molecules or cytokines known
in the art.
The term "bispecific single chain antibody construct" relates to a
construct comprising one domain consisting of variable regions (or
parts thereof) as defined above, capable of specifically
interacting with/binding to human CD3 and comprising a second
domain consisting of variable regions (or parts thereof as defined
above, capable of specifically interacting with/binding to human
CD19.
Said binding/interaction is also understood to define a "specific
recognition". The term "specifically recognizing" means in
accordance with this invention that the antibody molecule is
capable of specifically interacting with and/or binding to at least
two amino acids of each of the human target molecule as defined
herein. Said term relates to the specificity of the antibody
molecule, i.e. to its ability to discriminate between the specific
regions of the human target molecule as defined herein. The
specific interaction of the antigen-interaction-site with its
specific antigen may result in an initiation of a signal, e.g. due
to the induction of a change of the conformation of the antigen, an
oligomerization of the antigen, etc. Further, said binding may be
exemplified by the specificity of a "key-lock-principle". Thus,
specific motifs in the amino acid sequence of the
antigen-interaction-site and the antigen bind to each other as a
result of their primary, secondary or tertiary structure as well as
the result of secondary modifications of said structure. The
specific interaction of the antigen-interaction-site with its
specific antigen may result as well in a simple binding of said
site to the antigen.
The term "specific interaction" as used in accordance with the
present invention means that the bispecific single chain construct
does not or essentially does not cross-react with (poly)peptides of
similar structures. Cross-reactivity of a panel of bispecific
single chain construct under investigation may be tested, for
example, by assessing binding of said panel of bispecific single
chain construct under conventional conditions (see, e.g., Harlow
and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, 1988 and Using Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, 1999) to the (poly)peptide of
interest as well as to a number of more or less (structurally
and/or functionally) closely related (poly)peptides. Only those
antibodies that bind to the (poly)peptide/protein of interest but
do not or do not essentially bind to any of the other
(poly)peptides are considered specific for the
(poly)peptide/protein of interest.
Examples for the specific interaction of an
antigen-interaction-site with a specific antigen comprise the
specificity of a ligand for its receptor. Said definition
particularly comprises the interaction of ligands which induce a
signal upon binding to its specific receptor. Examples for
corresponding ligands comprise cytokines which interact/bind
with/to its specific cytokine-receptors. Also particularly
comprised by said definition is the binding of an
antigen-interaction-site to antigens like antigens of the selectin
family, integrins and of the family of growth factors like EGF. An
other example for said interaction, which is also particularly
comprised by said definition, is the interaction of an antigenic
determinant (epitope) with the antigenic binding site of an
antibody.
The term "binding to/interacting with" may also relate to a
conformational epitope, a structural epitope or a discountinuous
epitope consisting of two regions of the human target molecules or
parts thereof. In context of this invention, a conformational
epitope is defined by two or more discrete amino acid sequences
separated in the primary sequence which come together on the
surface of the molecule when the polypeptide folds to the native
protein (Sela, (1969) Science 166, 1365 and Laver, (1990) Cell 61,
553-6).
The term "discontinuous epitope" means in context of the invention
non-linear epitopes that are assembled from residues from distant
portions of the polypeptide chain. These residues come together on
the surface of the molecule when the polypeptide chain folds into a
three-dimensional structure to constitute a
conformational/structural epitope.
According to the present invention the term "variable region" used
in the context with Ig-derived antigen-interaction comprises
fragments and derivatives of (poly)peptides which at least comprise
one CDR derived from an antibody, antibody fragment or derivative
thereof. It is envisaged by the invention, that said at least one
CDR is preferably a CDR3, more preferably the CDR3 of the heavy
chain of an antibody (CDR-H3). However, other antibody derived CDRs
are also particularly comprised by the term "variable region"
The "specific binding" of an antibody is characterized primarily by
two parameters: a qualitative parameter (the binding epitope, or
where the antibody binds) and a quantitative parameter (the binding
affinity, or how strongly it binds where it does). Which epitope is
bound by an antibody can advantageously be determined by e.g. known
FACS methodology, peptide-spot epitope mapping, mass spectroscopy.
The strength of antibody binding to a particular epitope may be
advantageously be determined by e.g. known BIAcore and/or ELISA
methodologies. A combination of such techniques allows the
calculation of a signal:noise ratio as a representative measure of
binding specificity. In such a signal:noise ratio, the signal
represents the strength of antibody binding to the epitope of
interest, whereas the noise represents the strength of antibody
binding to other, non-related epitopes differing from the epitope
of interest. In general, any time an antibody binds more frequently
and/or strongly to one epitope than another epitope, such antibody
may be said to bind the former epitope specifically. Preferably, a
signal:noise ratio for an epitope of interest which is about
50-fold higher than for other epitopes different from the epitope
of interest may be taken as an indication that the antibody
evaluated binds the epitope of interest in a specific manner, i.e.
is a "specific binder".
As will be detailed below, a part of a variable region may be at
least one CDR ("Complementary determining region"), most preferably
at least the CDR3 region. Said two domains/regions in the single
chain antibody construct are preferably covalently connected to one
another as a single chain. This connection can be effected either
directly (domain1 directed against CD3--domain2 directed against CD
19 or domain1 directed against CD19--domain2 directed against CD3)
or through an additional polypeptide linker sequence
(domain1--linker sequence--domain2). In the event that a linker is
used, this linker is preferably of a length and sequence sufficient
to ensure that each of the first and second domains can,
independently from one another, retain their differential binding
specificities. Most preferably and as documented in the appended
examples, the "bispecific single chain antibody construct" to be
employed in the pharmaceutical composition of the invention is a
bispecific single chain Fv (scFv). Bispecific single chain
molecules are known in the art and are described in WO 99/54440,
Mack, J. Immunol. (1997), 158, 3965-3970, Mack, PNAS, (1995), 92,
7021-7025, Kufer, Cancer Immunol. Immunother., (1997), 45, 193-197,
Loffler, Blood, (2000), 95, 6, 2098-2103, Bruhl, Immunol., (2001),
166, 2420-2426, Kipriyanov, J. Mol. Biol., (1999), 293,41-56.
The term "single-chain" as used in accordance with the present
invention means that said first and second domain of the bispecific
single chain construct are covalently linked, preferably in the
form of a co-linear amino acid sequence encodable by a single
nucleic acid molecule.
As pointed out above, CD19 denotes an antigen that is expressed in
the B lineage such as in the pro B cell and the mature B cell, it
is not shed, is uniformly expressed on all lymphoma cells, and is
absent from stem cells (Haagen (1992) loc.cit; Uckun (1988) PNAS
85, 8603-8607). CD3 denotes an antigen that is expressed on T-cells
as part of the multimolecular T-cell receptor complex and that
consists of at least three different chains CD3.epsilon.,
CD3.delta. and CD3.gamma.. Clustering of CD3 on T-cells, e.g., by
immobilized anti-CD3-antibodies, leads to T-cell activation similar
to the engagement of the T-cell receptor but independent from its
clone typical specificity. Actually, most anti-CD3-antibodies
recognize the CD3.epsilon.-chain.
Antibodies that specifically recognize CD19 or CD3 antigen are
described in the prior art, e.g., in Dubel (1994), J. Immunol.
Methods 175, 89-95; Traunecker (1991) EMBO J. 10, 3655-3699 or
Kipriyanov, (1998), loc.cit. Further illustrative examples are
listed below. Furthermore, antibodies directed against human CD3
and/or human CD19 can be generated by conventional methods known in
the art.
Here it was surprisingly found that bispecific single chain
constructs directed against human CD3 and human CD19 and comprising
variable regions (V.sub.H (corresponds to V.sub.H),V.sub.L
(corresponds to V.sub.L)) or parts thereof (e.g. CDRs) in the
format V.sub.H(CD19)-V.sub.L(CD19)-V.sub.H(CD3)-V.sub.L(CD3),
V.sub.H(CD3)-V.sub.L(CD3)-V.sub.H(CD19)-V.sub.L(CD19) or
V.sub.H(CD3)-V.sub.L(CD3)-V.sub.L(CD19)-V.sub.H(CD19) are
particularly useful as pharmaceutical compositions since these
constructs are advantageous over constructs of similar formats,
like V.sub.L(CD3)-V.sub.H(CD3)-V.sub.L(CD19)-V.sub.H(CD19),
V.sub.L(CD3)-V.sub.H(CD3)-V.sub.H(CD19)-V.sub.L(CD19),
V.sub.L(CD19)-V.sub.H(CD19)-V.sub.L(CD3)-V.sub.H(CD3) or
V.sub.H(CD19)-V.sub.L(CD19)-V.sub.L(CD3)-V.sub.H(CD3). The latter
four constructs/construct formats are characterized by less
advantageous cytotoxic activity as reflected by EC.sub.50 values
and/or less efficient or complete purifications as shown in the
appended examples. It was in particular surprising that the
anti-CD3 part of the single chain constructs to be employed in
accordance with the invention are highly bioactive in N- as well as
C-terminal position, whereas arrangements in
V.sub.H(CD3)-V.sub.L(CD3) are particularly preferred. The
constructs to be employed in the pharmaceutical composition of the
invention are characterized by advantageous production and
purification properties as well as by their high bioactivity, i.e.
their desired cytotoxic activity. The corresponding high
bioactivity is reflected by low to very low EC.sub.50 values as
determined in cytotoxicity tests. The term "EC.sub.50" corresponds,
in context of this invention, to EC.sub.50 values as determined
according to the methods known in the art and as illustrated in the
appended examples: a standard dose-response curve is defined by
four parameters: the baseline response (Bottom), the maximum
response (Top), the slope of dose-response increase, and the drug
concentration that elicits a response halfway between baseline and
maximum (EC.sub.50). EC.sub.50 is defined as the concentration of a
drug or molecule that elicits a response half way between the
baseline (Bottom) and maximum response (Top). The percentage of
cell lysis (i.e. cytotoxic activity) may be determined by, inter
alia, release assays disclosed herein above, for example, .sup.51Cr
release assays, LDH-release assays, calcein release assays and the
like. Most preferably, in the context of this invention
fluorochrome release assays are employed as illustrated in the
appended examples. Here, strong cytotoxic activity against
CD19-positive cells (experimentally for example NALM6 cells) of the
bispecific single chain constructs described herein relates to a
molecule comprising EC.sub.50 values </- (less or equal to) 500
pg/ml, more preferably </-400 pg/ml, even more preferably
</-300 pg/ml, even more preferably </-250 pg/ml, most
preferably </-200 pg/ml. Here, it was surprisingly found that
certain constructs having the formats
VH(CD19)-VL(CD19)-VH(CD3)-VL(CD3) and
VH(CD3)-VL(CD3)-VH(CD19)-VL(CD19) demonstrate advantageous
properties in addition to high cytotoxic activity which make these
constructs well-suited to inclusion in pharmaceutical compositions.
In contrast, other constructs such as
VH(CD19)-VL(CD19)-VL(CD3)-VH(CD3) are only very poorly
producible/isolatable making, for example the latter construct very
poorly suited to inclusion in pharmaceutical compositions.
In a preferred embodiment of the pharmaceutical composition of this
invention, the VH and VL regions of said CD3 specific domain are
derived from a CD3 specific antibody selected from the group
consisting of X35-3, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7,
YTH12.5, F111-409, CLB-T3.4.2, WT31, WT32, SPv-T3b, 11D8, XIII-141,
XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2
and F101.01. These CD3-specific antibodies are well known in the
art and, inter alia, described in Tunnacliffe (1989), Int. Immunol.
1, 546-550. In a more preferred embodiment, said VH and VL regions
of said CD3 specific domain are derived from OKT-3 (as defined and
described above) or TR-66. Even more preferred (and as illustrated
in the appended examples) said VH and VL regions are or are derived
from an antibody/antibody derivative specifically directed against
CD3 described by Traunecker (1991), EMBO J. 10, 3655-3659. In
accordance with this invention, said VH and VL regions are derived
from antibodies/antibody derivatives and the like which are capable
of specifically recognizing human CD3 epsilon in the context of
other TCR subunits, e.g. in mouse T cells transgenic for human CD3
epsilon. These transgenic mouse cells express human CD3 epsilon in
a native or near native conformation. Accordingly, the VH and VL
regions derived from a CD3-epsilon-specific antibody are most
preferred in accordance with this invention and said (parental)
antibodies should be capable of specifically binding epitopes
reflecting the native or near native structure or a conformational
epitope of human CD3 presented in context of the TCR complex. Such
antibodies have been classified by Tunnacliffe (1989) as "group II"
antibodies. Further classifications in Tunnacliffe (1989) comprise
the definition of "group I" and "group III" antibodies directed
against CD3. "Group I" antibodies, like UCHT1, recognize CD3
epsilon both expressed as recombinant protein as well as part of
the TCR on the cell surface. Therefore, "group I" antibodies are
highly specific for CD3 epsilon. In contrast, the herein preferred
"group II" antibodies recognize CD3 epsilon only in the native TCR
complex in association with other TCR subunits. Without being bound
by theory, it is speculated in context of this invention that in
"group II" antibodies, the TCR context is required for recognition
of CD3 epsilon. CD3 gamma and/or delta, being associated with
epsilon, are also involved in binding of "group II" antibodies. All
three subunits express immuno-tyrosine activation motifs (ITAMs)
which can be tyrosine phosphorylated by protein tyrosine kinases.
For this reason "group II" antibodies induce T cell signaling via
CD3 epsilon, gamma and delta, leading to a stronger signal compared
to "group I" antibodies selectively inducing T cell signaling via
CD3 epsilon. Yet, since for therapeutic applications induction of a
strong T cell signaling is desired, the VH (CD3)/VL (CD3)-regions
(or parts thereof) to be employed in the bispecific single chain
constructs comprised in the inventive pharmaceutical composition,
are preferably derived from antibodies directed against human CD3
and classified as "group II" by Tunnacliffe (1989), loc.cit.
Antibodies/antibody molecules/antibody derivatives directed against
human CD19 which provide for variable regions (V.sub.Hand V.sub.L)
to be employed in the bispecific single chain construct(s)
comprised in the inventive pharmaceutical composition are also well
known in the art and illustrated in the appended examples.
Preferred antibodies directed to human CD19 are: 4G7 (Meecker
(1984) Hybridoma 3, 305-20); B4 (Freedman (1987) Blood 70, 418-27;
B43 (Bejcek (1995) Cancer Res. 55, 2346-51); BU12 (Flavell (1995)
Br. J. Cancer 72, 1373-9); CLB-CD19 (De Rie (1989) Cell. Immunol.
118, 368-81); Leu-12 (MacKenzie (1987), J. Immunol. 139, 24-8);
SJ25-C1 (GenTrak, Plymouth Meeting, Pa.)
In a most preferred embodiment of the invention said V.sub.H(CD19)
and V.sub.L(CD19) regions (or parts, like CDRs, thereof) are
derived from the antibody provided by the HD37 hybridoma (Pezzutto
(1997), J. Immunol. 138, 2793-9).
As is well known, Fv, the minimum antibody fragment which contains
a complete antigen recognition and binding site, consists of a
dimer of one heavy and one light chain variable domain (VH and VL)
in non-covalent association. In this configuration corresponding to
the one found in native antibodies, the three CDRs of each variable
domain interact to define an antigen binding site on the surface of
the VH-VL dimer. Collectively, the six CDRs confer antigen binding
specificity to the antibody. Frameworks (FRs) flanking the CDRs
have a tertiary structure which is essentially conserved in native
immunoglobulins of species as diverse as human and mouse. These FRs
serve to hold the CDRs in their appropriate orientation. The
constant domains are not required for binding function, but may aid
in stabilizing VH-VL interaction.
It is also envisaged in context of the present invention that the
bispecific antibody constructs provided in the pharmaceutical
composition of the invention are further modified. In particular,
it is envisaged that the bispecific single chain antibody construct
in the format
V.sub.H(CD19)-V.sub.L(CD19)-V.sub.H(CD3)-V.sub.L(CD3),
V.sub.H(CD3)-V.sub.L(CD3)-V.sub.H(CD19)-V.sub.L(CD19) or
V.sub.H(CD3)-V.sub.L(CD3)-V.sub.L(CD19)-V.sub.H(CD19) as defined
herein are deimmunized. Most preferably, at least the CD3-binding
portion is deimmunized. Deimmunization entails carrying out
substitutions of amino acids within potential T cell epitopes.
It is envisaged and preferred that the pharmaceutical composition
of the invention, comprises a bispecific single chain antibody
construct in the format
V.sub.H(CD19)-V.sub.L(CD19)-V.sub.H(CD3)-V.sub.L(CD3),
V.sub.H(CD3)-V.sub.L(CD3)-V.sub.H(CD19)-V.sub.L(CD19) or
V.sub.H(CD3)-V.sub.L(CD3)-V.sub.L(CD19)-V.sub.H(CD19) as defined
above, wherein said V.sub.Hregion comprises at least one CDR3
region (CDR-H3 or CDR-3 of V.sub.H) comprising the amino acid
sequence: SEQ ID NO. 54 or 77.
The term "CDR-region" as used herein denotes the "complementary
determining region" of an antibody molecule. Accordingly, the term
"CDR-3 region", synonymous with the term "CDR3 region", relates to
the "complementary determining region 3" of an antibody
molecule/antibody construct. The same applies, mutatis mutandis,
for corresponding CDR-2 and CDR-1 regions. It is envisaged and
preferred that the bispecific single chain construct comprised in
the pharmaceutical composition of the present invention does not
only comprise CDR-3 regions, but also comprises CDR-1 or CDR-2
region(s) of variable regions/variable domains (VH/VL) of
antibodies/antibody molecules directed against human CD3 and human
CD19. Most preferably, the said molecule comprises at least one
CDR-3 region of a VH and at least one CDR-3 region of an VL-domain
of an antibody directed against CD3 as well as at least one CDR-3
region of an VH and at least one CDR-3 region of a VL-domain of an
antibody directed against CD19. Most preferably, the bispecific
single chain construct of the inventive pharmaceutical composition
comprises in addition at least one further CDR-1 region and/or at
least one further CDR-2 region in the VH and VL domains defined
herein. Accordingly, the bispecific single chain construct defined
herein may comprise CDR-1, CDR-2, CDR-3 region of VL as well as
CDR-1, CDR-2, CDR-3 region of VH of an antibody/antibody molecule
directed against human CD3, preferably human CD3 epsilon, and
comprises, in addition, CDR-1, CDR-2, CDR-3 region of VL as well as
CDR-1, CDR-2, CDR-3 region of VH of an antibody/antibody molecule
directed against human CD19.
Preferably, said VH (CD3) region comprises at least one CDR2 region
comprising the amino acid sequence: SEQ ID NO. 53 or 76. It is also
envisaged that said VH (CD3) region comprises at least one CDR1
region comprising the amino acid sequence: SEQ ID NO. 52 or 75.
The VL (CD3) region comprises, preferably, at least one CDR3 region
comprising the amino acid sequence: SEQ ID NO. 57 or 74. The VL
(CD3) may comprise at least one CDR2 region comprising the amino
acid sequence: SEQ ID NO. 56 or 73. The VL (CD3) may also comprise
at least one CDR1 region comprising the amino acid sequence: SEQ ID
NO. 55 or 72.
As mentioned herein above, the constructs comprised in the
inventive pharmaceutical composition comprise at least one CDR-3 of
a VH-region of an antibody directed against human CD3, at least one
CDR-3 of a VL-region of an antibody directed against human CD3, at
least one CDR-3 of a VH-region of an antibody directed against
human CD19 and at least one CDR-3 of a VL-region of an antibody
directed against human CD19. However, in a most preferred
embodiment and as illustrated in the appended examples, the
bispecific single chain constructs comprised in the inventive
pharmaceutical composition comprise VH and VL regions which
comprise not only CDR-3 but also CDR1 and/or CDR2 regions. In
particular, CDR-regions, preferably CDR1 regions, more preferably
CDR1 regions and CDR2 regions, most preferably CDR1 regions, CDR2
regions and CDR3 regions as defined herein may be employed to
generate further bispecific single chain constructs defined herein.
Most preferably the bispecific single chain constructs comprised in
the inventive pharmaceutical composition are derived from the
parental antibodies as disclosed herein and share, as disclosed
above, the CDR-3 domain of the VH-region and the CDR-3 domain of
the VL-region with said parental antibodies. Yet, it is also
envisaged that the bispecific single chain constructs comprised in
the inventive pharmaceutical composition also comprises modified
CDR regions. It is, e.g. envisaged that in particular CDR2 and/or
CDR1 regions (or frameworks or linkers between CDRs) are
deimmunized.
In a preferred embodiment of the invention the bispecific single
chain antibody construct comprised in the inventive pharmaceutical
composition comprises an amino acid sequence selected from the
group consisting of (a) an amino acid sequence as depicted in SEQ
ID NOs 2, 10 or 14; (b) an amino acid sequence encoded by a nucleic
acid sequence as shown in SEQ ID NOs 1, 9 or 13; (c) an amino acid
sequence encoded by a nucleic acid sequence hybridizing under
stringent conditions to the complementary nucleic acid sequence of
(b); and (d) an amino acid sequence encoded by a nucleic acid
sequence which is degenerate as a result of the genetic code to a
nucleotide sequence of (b).
The term "hybridizing" as used herein refers to
polynucleotides/nucleic acid sequences which are capable of
hybridizing to the polynucleotides encoding bispecific single chain
constructs as defined herein or parts thereof. Therefore, said
polynucleotides may be useful as probes in Northern or Southern
Blot analysis of RNA or DNA preparations, respectively, or can be
used as oligonucleotide primers in PCR analysis dependent on their
respective size. Preferably, said hybridizing polynucleotides
comprise at least 10, more preferably at least 15 nucleotides in
length while a hybridizing polynucleotide of the present invention
to be used as a probe preferably comprises at least 100, more
preferably at least 200, or most preferably at least 500
nucleotides in length.
It is well known in the art how to perform hybridization
experiments with nucleic acid molecules, i.e. the person skilled in
the art knows what hybridization conditions s/he has to use in
accordance with the present invention. Such hybridization
conditions are referred to in standard text books such as Molecular
Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (2001)
N.Y. Preferred in accordance with the present inventions are
polynucleotides which are capable of hybridizing to the
polynucleotides of the invention or parts thereof, under stringent
hybridization conditions.
"Stringent hybridization conditions" refer, i.e. to an overnight
incubation at 42.degree. C. in a solution comprising 50% formamide,
5.times.SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium
phosphate (pH 7.6), 5.times. Denhardt's solution, 10% dextran
sulfate, and 20 .mu.g/ml denatured, sheared salmon sperm DNA,
followed by washing the filters in 0.1.times.SSC at about
65.degree. C. Also contemplated are nucleic acid molecules that
hybridize to the polynucleotides of the invention at lower
stringency hybridization conditions. Changes in the stringency of
hybridization and signal detection are primarily accomplished
through the manipulation of formamide concentration (lower
percentages of formamide result in lowered stringency); salt
conditions, or temperature. For example, lower stringency
conditions include an overnight incubation at 37.degree. C. in a
solution comprising 6.times.SSPE (20.times.SSPE=3M NaCl; 0.2M
NaH2po4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 .mu.g/ml
salmon sperm blocking DNA; followed by washes at 50.degree. C. with
1.times.SSPE, 0.1% SDS. In addition, to achieve even lower
stringency, washes performed following stringent hybridization can
be done at higher salt concentrations (e.g. 5.times.SSC). It is of
note that variations in the above conditions may be accomplished
through the inclusion and/or substitution of alternate blocking
reagents used to suppress background in hybridization experiments.
Typical blocking reagents include Denhardt's reagent, BLOTTO,
heparin, denatured salmon sperm DNA, and commercially available
proprietary formulations. The inclusion of specific blocking
reagents may require modification of the hybridization conditions
described above, due to problems with compatibility.
As mentioned above, the said variable domains comprised in the
herein described bispecific single chain constructs are connected
by additional linker sequences. The term "peptide linker" defines
in accordance with the present invention an amino acid sequence by
which the amino acid sequences of the first domain and the second
domain of the monomer of the trimeric polypeptide construct of the
invention are linked with each other. An essential technical
feature of such peptide linker is that said peptide linker does not
comprise any polymerization activity. A particularly preferred
peptide linker is characterized by the amino acid sequence
Gly-Gly-Gly-Gly-Ser, i.e. Gly.sub.4Ser, or polymers thereof, i.e.
(Gly.sub.4Ser)x, where x is an integer 1 or greater. The
characteristics of said peptide linker, which comprise the absence
of the promotion of secondary structures are known in the art and
described e.g. in Dall'Acqua et al. (Biochem. (1998) 37,
9266-9273), Cheadle et al. (Mol Immunol (1992) 29, 21-30) and Raag
and Whitlow (FASEB (1995) 9(1), 73-80). Also particularly preferred
are peptide linkers which comprise fewer amino acid residues. An
envisaged peptide linker with less than 5 amino acids comprises 4,
3, 2 or one amino acid(s) wherein Gly-rich linkers are preferred. A
particularly preferred "single" amino acid in context of said
"peptide linker" is Gly. Accordingly, said peptide linker may
consist of the single amino acid Gly. Furthermore, peptide linkers
which also do not promote any secondary structures are preferred.
The linkage of said domains to each other can be provided by, e.g.
genetic engineering, as described in the examples. Methods for
preparing fused and operatively linked bispecific single chain
constructs and expressing them in mammalian cells or bacteria are
well-known in the art (e.g. WO 99/54440 or Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 2001).
The present invention also provides for a pharmaceutical
composition comprising a nucleic acid sequence encoding a
bispecific single chain antibody construct as defined above, i.e. a
bispecific construct in the format
VH(CD19)-VL(CD19)-VH(CD3)-VL(CD3), VH(CD3)-VL(CD3)-VH(CD19)VL(CD19)
or VH(CD3)-VL(CD3)-VL(CD19)-VH(CD19). Of these, nucleic acid
sequences encoding bispecific constructs of the formats
VH(CD19)-VL(CD19)-VH(CD3)-VL(CD3) and
VH(CD3)-VL(CD3)-VH(CD19)-VL(CD19) are each especially advantageous
for inclusion in such pharmaceutical compositions. In contrast, a
nucleic acid sequence encoding a bispecific construct of, for
example, the format VH(CD19)-VL(CD19)-VL(CD3)-VH(CD3) is very
poorly suited for inclusion in pharmaceutical compositions, the
latter being very poorly producible/isolatable.
Said nucleic acid molecule may be a naturally occurring nucleic
acid molecule as well as a recombinant nucleic acid molecule. The
nucleic acid molecule of the invention may, therefore, be of
natural origin, synthetic or semi-synthetic. It may comprise DNA,
RNA as well as PNA and it may be a hybrid thereof.
It is evident to the person skilled in the art that regulatory
sequences may be added to the nucleic acid molecule of the
invention. For example, promoters, transcriptional enhancers and/or
sequences which allow for induced expression of the polynucleotide
of the invention may be employed. A suitable inducible system is
for example tetracycline-regulated gene expression as described,
e.g., by Gossen and Bujard (Proc. Natl. Acad. Sci. USA 89 (1992),
5547-5551) and Gossen et al. (Trends Biotech. 12 (1994), 58-62), or
a dexamethasone-inducible gene expression system as described, e.g.
by Crook (1989) EMBO J. 8, 513-519.
Furthermore, it is envisaged for further purposes that nucleic acid
molecules may contain, for example, thioester bonds and/or
nucleotide analogues. Said modifications may be useful for the
stabilization of the nucleic acid molecule against endo- and/or
exonucleases in the cell. Said nucleic acid molecules may be
transcribed by an appropriate vector containing a chimeric gene
which allows for the transcription of said nucleic acid molecule in
the cell. In this respect, it is also to be understood that the
polynucleotide of the invention can be used for "gene targeting" or
"gene therapeutic" approaches. In another embodiment said nucleic
acid molecules are labeled. Methods for the detection of nucleic
acids are well known in the art, e.g., Southern and Northern
blotting, PCR or primer extension. This embodiment may be useful
for screening methods for verifying successful introduction of the
nucleic acid molecules described above during gene therapy
approaches.
Said nucleic acid molecule(s) may be a recombinantly produced
chimeric nucleic acid molecule comprising any of the aforementioned
nucleic acid molecules either alone or in combination. Preferably,
the nucleic acid molecule is part of a vector.
The present invention therefore also relates to a pharmaceutical
composition comprising a vector comprising the nucleic acid
molecule described in the present invention.
The vector of the present invention may be, e.g., a plasmid,
cosmid, virus, bacteriophage or another vector used e.g.
conventionally in genetic engineering, and may comprise further
genes such as marker genes which allow for the selection of said
vector in a suitable host cell and under suitable conditions.
Furthermore, the vector to be employed in the generation of the
bispecific single chain constructs described herein or to be
employed in a pharmaceutical composition of the present invention
may, in addition to the nucleic acid sequences of the invention,
comprise expression control elements, allowing proper expression of
the coding regions in suitable hosts. Such control elements are
known to the artisan and may include a promoter, a splice cassette,
translation initiation codon, translation and insertion site for
introducing an insert into the vector. Preferably, said nucleic
acid molecule is operatively linked to said expression control
sequences allowing expression in eukaryotic or prokaryotic
cells.
Control elements ensuring expression in eukaryotic and prokaryotic
cells are well known to those skilled in the art. As mentioned
herein above, they usually comprise regulatory sequences ensuring
initiation of transcription and optionally poly-A signals ensuring
termination of transcription and stabilization of the transcript.
Additional regulatory elements may include transcriptional as well
as translational enhancers, and/or naturally-associated or
heterologous promoter regions. Possible regulatory elements
permitting expression in for example mammalian host cells comprise
the CMV-HSV thymidine kinase promoter, SV40, RSV-promoter (Rous
Sarcoma Virus), human elongation factor 1.alpha.-promoter, the
glucocorticoid-inducible MMTV-promoter (Moloney Mouse Tumor Virus),
metallothionein- or tetracyclin-inducible promoters, or enhancers,
like CMV enhancer or SV40-enhancer. For expression in white blood
cells, it is envisaged that specific promoters can be employed.
Said promoters are known in the art and, inter alia, described or
mentioned in Hendon (2002), Clin. Immunol. 103, 145-153; Chinnosamy
(2000) Blood 96, 1309-1316; Zhang (2003) J. Acq. Immun. Def. Synd.
245-254; Kaiser (2003) Science 299, 495; Hacein-Bay (2002) Int. J.
Hemat. 76, 295-298; Hacein-Bay (2002) New Eng. J. Med. 346,
1185-1193; Ainti (2002) Science 296, 2410-2413. For the expression
in prokaryotic cells, a multitude of promoters including, for
example, the tac-lac-promoter or the trp promoter, has been
described. Besides elements which are responsible for the
initiation of transcription such regulatory elements may also
comprise transcription termination signals, such as SV40-poly-A
site or the tk-poly-A site, downstream of the polynucleotide. In
this context, suitable expression vectors are known in the art such
as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc/CMV,
pcDNA1, pcDNA3 (In-vitrogene), pSPORT1 (GIBCO BRL), pX (Pagano
(1992) Science 255, 1144-1147), yeast two-hybrid vectors, such as
pEG202 and dpJG4-5 (Gyuris (1995) Cell 75, 791-803), or prokaryotic
expression vectors, such as lambda gt11 or pGEX
(Amersham-Pharmacia). Beside the nucleic acid molecules coding for
the bispecific single chain constructs described herein, the vector
may further comprise nucleic acid sequences encoding for secretion
signals. Such sequences are well known to the person skilled in the
art. Furthermore, depending on the expression system used, leader
sequences capable of directing the peptides of the invention to a
cellular compartment may be added to the coding sequence of the
nucleic acid molecules of the invention and are well known in the
art. The leader sequence(s) is (are) assembled in appropriate phase
with translation, initiation and termination sequences, and
preferably, a leader sequence capable of directing secretion of
translated protein, or a protein thereof, into the periplasmic
space or extracellular medium. Optionally, the heterologous
sequence can encode a fusion protein including an C- or N-terminal
identification peptide imparting desired characteristics, e.g.,
stabilization or simplified purification of expressed recombinant
product. Once the vector has been incorporated into the appropriate
host, the host is maintained under conditions suitable for high
level expression of the nucleotide sequences, and, as desired, the
collection and purification of the bispecific single chain
constructs described herein may follow. The invention also relates,
accordingly, to hosts/host cells which comprise a vector as defined
herein. Such hosts may be useful for in processes for obtaining
bispecifc single chain constructs comprised in the pharmaceutical
composition of the invention as well as directly in
medical/pharmaceutical settings. Said host cells may also comprise
transduced or transfected white blood cells, such as lymphocyte
cells, preferably adult cells. Such host cells may be useful in
transplantation therapies.
Furthermore, the vector as well as the nucleic acid molecule
described herein may be employed in gene therapy approaches. Gene
therapy, which is based on introducing therapeutic genes into cells
by ex-vivo or in-vivo techniques is one of the most important
applications of gene transfer. Suitable vectors, methods or
gene-delivering systems for in-vitro or in-vivo gene therapy are
described in the literature and are known to the person skilled in
the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539;
Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256
(1992), 808-813; Isner, Lancet 348 (1996), 370-374; Muhlhauser,
Circ. Res. 77 (1995), 1077-1086; Onodua, Blood 91 (1998), 30-36;
Verzeletti, Hum. Gene Ther. 9 (1998), 2243-2251; Verma, Nature 389
(1997), 239-242; Anderson, Nature 392 (Supp. 1998), 25-30; Wang,
Gene Therapy 4 (1997), 393-400; Wang, Nature Medicine 2 (1996),
714-716; WO 94/29469; WO 97/00957; U.S. Pat. No. 5,580,859; U.S.
Pat. No. 5,589,466; U.S. Pat. No. 4,394,448 or Schaper, Current
Opinion in Biotechnology 7 (1996), 635-640, and references cited
therein. In particular, said vectors and/or gene delivery systems
are also described in gene therapy approaches in blood,
lymphocytes, bone marrow and corresponding stem cells; see, e.g.
Hendon (2002), Clin. Immunol. 103, 145-153; Chinnosamy (2000) Blood
96, 1309-1316; Zhang (2003) J. Acq. Immun. Def. Synd. 245-254;
Kaiser (2003) Science 299, 495; Hacein-Bay (2002) Int. J. Hemat.
76, 295-298; Hacein-Bay (2002) New Eng. J. Med. 346, 1185-1193;
Ainti (2002) Science 296, 2410-2413. The nucleic acid molecules and
vectors comprised in the pharmaceutical composition of the
invention may be designed for direct introduction or for
introduction via liposomes, viral vectors (e.g. adenoviral,
retroviral), electroporation, ballistic (e.g. gene gun) or other
delivery systems into the cell. Additionally, a baculoviral system
can be used as a eukaryotic expression system in insect cells for
the nucleic acid molecules of the invention. The introduction and
gene therapeutic approach should, preferably, lead to the
expression of a functional bispecific single chain construct as
defined herein, whereby said expressed antibody molecule is
particularly useful in the treatment, amelioration and/or
prevention of B-cell related malignancies as defined herein.
Preferably, the expression control sequences will be eukaryotic
promoter systems in vectors capable of transforming of transfecting
eukaryotic host cells, but control sequences for prokaryotic hosts
may also be used. Once the vector has been incorporated into the
appropriate host, the host is maintained under conditions suitable
for high level expression of the nucleotide sequences, and as
desired, the collection and purification of the bispecific single
chain constructs may follow; see, e.g., the appended examples.
Therefore, in further embodiments of the invention, a
pharmaceutical composition is provided which comprising a vector
encoding a bispecific single chain construct in the format
V.sub.H(CD19)-V.sub.L(CD19)-V.sub.H(CD3)-V.sub.L(CD3),
V.sub.H(CD3)-V.sub.L(CD3)-V.sub.H(CD19)-V.sub.L(CD19) or
V.sub.H(CD3)-V.sub.L(CD3)-V.sub.L(CD19)-V.sub.H(CD19) or a host
transformed or transfected with said vector.
The pharmaceutical composition of the invention may also comprise a
proteinaceous compound capable of providing an additional
activation signal for immune effector cells. Such compounds may
comprise, but are not limited to CD28 engagers, ICOS engagers,
4-1BB engagers, OX40 engagers, CD27 engagers, CD30 engagers, NKG2D
engagers, IL2-R engagers or IL12-R engagers. In the light of the
present invention, said "proteinaceous compounds" providing an
activation signal for immune effector cells" may be, e.g. a further
primary activation signal, or costimulatory (second) signal or any
other accessory (third) activation signal. Examples are a TCR or
TCR-like signal. Preferred formats of proteinaceous compounds
comprise additional bispecific antibodies and fragments or
derivatives thereof, e.g. bispecific scFv. Proteinaceous compounds
can comprise, but are not limited to, scFv fragments specific for
4-1BB, OX 40, CD27, CD70 or the receptors for B7-RP 1, B7-H3 as
well as scFv fragments specific for the T cell receptor or
superantigens. Superantigens directly bind to certain subfamilies
of T cell receptor variable regions in an MHC-independent manner
thus mediating the primary T cell activation signal. The
proteinaceous compound may also provide an activation signal for an
immune effector cell which is a non-T cell. Examples for immune
effector cells which are non-T cells comprise, inter alia, NK
cells.
In a further embodiment of the present invention, a process for the
production of a pharmaceutical composition of the invention is
provided, said process comprises culturing a host defined above
under conditions allowing the expression of the bispecific single
chain antibody construct as defined herein and recovering the
produced bispecific single chain antibody construct from the
culture. The corresponding process is illustrated in the appended
examples.
In a most preferred embodiment, the invention relates to the use of
a bispecific single chain antibody construct, a nucleic acid
sequence, a vector and/or a host as defined herein for the
preparation of a pharmaceutical composition for the prevention,
treatment or amelioration of a proliferative disease, a mimimal
residual cancer, a tumorous disease, an inflammatory disease, an
immunological disorder, an autoimmune disease, an infectious
disease, a viral disease, allergic reactions, parasitic reactions,
graft-versus-host diseases host-versus-graft diseases or B-cell
malignancies, wherein said pharmaceutical composition optionally
further comprises a proteinaceous compound capable of providing an
activation signal for immune effector cells.
Accordingly, a method for the prevention, treatment or amelioration
of a proliferative disease, a mimimal residual cancer, a tumorous
disease, an inflammatory disease, an immunological disorder, an
autoimmune disease, an infectious disease, viral disease, allergic
reactions, parasitic reactions, graft-versus-host diseases,
host-versus-graft diseases, or B-cell malignancies is provided,
whereby said method comprises the step of administering to a
subject in need of such a prevention, treatment or amelioration a
pharmaceutical composition of the invention. Most preferably said
subject is a human.
The tumorous disease to be treated with the pharmaceutical
composition of the invention may be a minimal residual cancer, for
example, a minimal residual lymphoma or leukemia.
The autoimmune disease to be treated with the pharmaceutical
composition of the invention may be in inflammatory autoimmune
disease, for example, rheumatoid arthritis.
In accordance with this invention, it is also envisaged that a
bispecific single chain antibody construct, a nucleic acid
sequence, a vector and/or a host as described herein is/are used
for the preparation of a pharmaceutical composition for depletion
of B-cells.
The B cell malignancy to be treated with the pharmaceutical
composition of the invention is in a most preferred embodiment
non-Hodgkin lymphoma, B-cell leukemias or Hodgkin lymphoma.
Accordingly, the present invention provides for a method for the
treatment of B-cell malignancies, B-cell mediated autoimmune
diseases or the depletion of B-cells and/or for a method delaying a
pathological condition which is caused by B-cell disorders
comprising administering the pharmaceutical composition of the
invention into a mammal, preferably a human, affected by said
malignancies, disease and/or pathological condition.
Finally, the invention provides for a kit comprising a bispecific
single chain antibody construct, a nucleic acid sequence, a vector
and/or a host as defined above. Said kit is particularly useful in
the preparation of the pharmaceutical composition of the present
invention and may, inter alia, consist of a container useful for
injections or infusions. Advantageously, the kit of the present
invention further comprises, optionally (a) buffer(s), storage
solutions and/or remaining reagents or materials required for the
conduct of medical or scientific purposes. Furthermore, parts of
the kit of the invention can be packaged individually in vials or
bottles or in combination in containers or multicontainer units.
The kit of the present invention may be advantageously used, inter
alia, for carrying out the method of the invention and could be
employed in a variety of applications referred herein, e.g., as
research tools or medical tools. The manufacture of the kits
preferably follows standard procedures which are known to the
person skilled in the art.
These and other embodiments are disclosed and encompassed by the
description and. Examples of the present invention. Further
literature concerning any one of the antibodies, methods, uses and
compounds to be employed in accordance with the present invention
may be retrieved from public libraries and databases, using for
example electronic devices. For example, the public database
"Medline", available on the Internet, may be utilized, for example
under http://www.ncbi.nlm.nih.gov/PubMed/medline.html. Further
databases and addresses, such as http://www.ncbi.nlm.nih.gov/,
http://www.infobiogen.fr/,
http://www.fmi.ch/biology/research_tools.html,
http://www.tigr.org/, are known to the person skilled in the art
and can also be obtained using, e.g., http://www.lycos.com.
The figures show:
FIG. 1A: Schematic composition of VL/VH domain arrangements in
anti-CD-19/ anti-CD3 single chain bispecific antibodies showing the
binding sites of PCR primers. A1, A2, B1and B2 denote the
positions, from the N-terminus to the C-terminus of the various
V-regions used in constructing the anti-CD19/anti-CD3 single chain
bispecific antibodies. (G.sub.4S.sub.1) and (G.sub.4S.sub.1).sub.2
linkers disclosed as SEQ ID NOS: 71 and 83, respectively.
FIG. 1B: Schematic composition of VL/VH domain arrangements in
anti-CD19/anti-CD3 single chain bispecific antibodies showing the
recognition site of restriction enzymes (L=Leader peptide). A1, A2,
B1 and B2 denote the positions, from the N-terminus to the
C-terminus of the various V-regions used in constructing the
anti-CD19/anti-CD3 single chain bispecific antibodies.
(G.sub.4S.sub.1) and (G.sub.4S.sub.1).sub.3 linkers disclosed as
SEQ ID NOS: 71 and 69, respectively.
FIG. 2: Bispecific single chain antibody elution pattern from a
Zn-chelating Fractogel.RTM. column (IMAC) at 280 nm. The bottom
line showing a first, minor step at 600 ml retention time and a
second, major step at 700 ml indicates the theoretical gradient of
elution buffer containing 0.5 M imidazole. High adsorption at 280
nm from 100-500 ml retention time was due to non-bound protein in
the column flow through. Protein from the elution peak at 670.05 ml
retention time was used for further purification.
FIG. 3: Bispecific single chain antibody elution pattern from a
Sephadex S200 gel filtration column at 280 nm. The protein peak at
81.04 ml retention time containing bispecific antibodies against
CD3 and CD19 corresponds to a molecular weight of 52 kD. Fractions
were collected from 50-110 ml retention time and were indicated
with black arrows numbered from 5-35.
FIG. 4: Representative SDS-PAGE analysis of protein fractions of
bispecific single chain antibodies. Lane M: Molecular weight marker
Lane 1: cell culture supernatant; lane 2: IMAC flow-through; lane
3: IMAC eluate; lane 4: purified antibody against CD19 and CD3
obtained from gel filtration (Sephadex 200).
FIG. 5: Representative western blot analysis of purified bispecific
single chain antibody fractions. Western blot analysis of purified
bispecific protein was performed with antibodies directed against
the HisTag (PentaHis, Qiagen) and goat anti mouse Ig labelled with
alkaline phosphatase. Lane 1: cell culture supernatant; lane 2:
IMAC flow-through; lane 3: IMAC eluate; lane 4: purified antibody
against CD19 and CD3 obtained from gel filtration (Sephadex
200).
FIG. 6A: Binding data for the anti-CD19 (VL/VH).times.anti-CD3
(VH/VL) construct as measured by FACS analysis on Nalm 6 (CD19+)
and Jurkat (CD3+) cells. The left peak is the control; the right
peak is the measurement of the fluorescence shift for the binding
specificity of interest. A shift to the right indicates binding of
the construct to CD19 or CD3, respectively. Arrangement of VH and
VL domains is indicated from N to C terminus (N.fwdarw.C).
FIG. 6B: Binding data for the anti-CD19 (VH/VL).times.anti-CD3
(VH/VL) construct as measured by FACS analysis on Nalm 6 (CD19+)
and Jurkat (CD3+) cells. The left peak is the control; the right
peak is the meacurement of the fluorescence shift for the binding
specificity of interest. A shift to the right indicates binding of
the construct to CD19 or CD3, respectively. Arrangement of VH and
VL domains is indicated from N to C terminus (N.fwdarw.C).
FIG. 6C: Binding data for the anti-CD19 (VL/VH).times.anti-CD3
(VL/VH) construct as measured by FACS analysis on Nalm 6 (CD19+)
and Jurkat (CD3+) cells. The left peak is the control; the right
peak is the meacurement of the fluorescence shift for the binding
specificity of interest. A shift to the right indicates binding of
the construct to CD19 or CD3, respectively. Arrangement of VH and
VL domains is indicated from N to C terminus (N.fwdarw.C).
FIG. 6D: Binding data for the anti-CD3 (VH/VL).times.anti-CD19
(VH/VL) construct as measured by FACS analysis on Nalm 6 (CD19+)
and Jurkat (CD3+) cells. The left peak is the control; the right
peak is the meacurement of the fluorescence shift for the binding
specificity of interest. A shift to the right indicates binding of
the construct to CD19 or CD3, respectively. Arrangement of VH and
VL domains is indicated from N to C terminus (N.fwdarw.C).
FIG. 6E: Binding data for the anti-CD3 (VL/VH).times.anti-CD19
(VL/VH) construct as measured by FACS analysis on Nalm 6 (CD19+)
and Jurkat (CD3+) cells. The left peak is the control; the right
peak is the meacurement of the fluorescence shift for the binding
specificity of interest. A shift to the right indicates binding of
the construct to CD19 or CD3, respectively. Arrangement of VH and
VL domains is indicated from N to C terminus (N.fwdarw.C).
FIG. 6F: Binding data for the anti-CD3 (VH/VL).times.anti-CD19
(VL/VH) construct as measured by FACS analysis on Nalm 6 (CD19+)
and Jurkat (CD3+) cells. The left peak is the control; the right
peak is the meacurement of the fluorescence shift for the binding
specificity of interest. A shift to the right indicates binding of
the construct to CD19 or CD3, respectively. Arrangement of VH and
VL domains is indicated from N to C terminus (N.fwdarw.C).
FIG. 7: Cytotoxicity data for selected domain-rearranged
anti-CD3/anti-CD19 constructs. CB15 T cell clone and NALM6 cells
were used in an E:T ratio of 1:10. NALM6 target cells were labelled
with calcein. Calcein release after cell lysis was determined by
FACS analysis.
FIG. 8: Binding of the 145-2C11 antibody to the recombinant,
purified extracellular domain of the murine CD3 epsilon chain in
ELISA. The ELISA was performed as described in Example 5, paragraph
1. The graph depicts absorption values for antigen preparation or
an irrelevant antigen binding to the coated 145-2C11 antibody.
Samples were done in 1:5, 1:25 and 1:125 dilution.
FIG. 9: FACS binding-analysis of the 145-2C11 antibody on Jurkat
cells transfected with the murine CD3 epsilon chain surface
antigen. The FACS staining was performed as described in Example 5,
paragraph 2. The filled histogram represents cells incubated with
the isotype control. The open histogram shows cells incubated with
the 145-2C11 antibody.
FIG. 10: FACS binding-analysis of the 145-2C11 antibody on
untransfected Jurkat cells. The FACS staining was performed as
described in Example 5, paragraph 2. The filled histogram
represents cells incubated with the isotype control. The open
histogram, superimposed on the filled histogram, represents cells
incubated with the 145-2C11 antibody. 145-2C11 did not bind to
Jurkat cells.
FIG. 11: FACS binding-analysis of the 145-2C11 antibody on CTLL2
cells. The FACS staining was performed as described in Example 5,
paragraph 3. The filled histogram represents cells incubated with
the isotype control. The open histogram indicates that the 145-2C11
antibody bound to CTLL2 cells.
The invention will now be described by reference to the following
examples which are merely illustrative and are not to be construed
as a limitation of the invention's scope.
EXAMPLE 1
Construction of CD19.times.CD3 and CD3.times.CD19 Single Chain
Bispecific Antibodies Comprising Various Domain Rearrangements
Generally, bispecific single antibody chain molecules, each
comprising a domain with binding specificity for the human CD3
antigen as well as a domain with binding specificity for the human
CD19 antigen, were designed as set out in the following Table
1:
TABLE-US-00001 TABLE 1 Formats of bispecific single antibody chain
molecules comprising anti-CD3 and anti-CD19 specificities SEQ ID
Construct Nos Formats of protein constructs Number (nuc/prot) (N
.fwdarw. C) 1 29/30 VL(CD19)-VH(CD19)-VH(CD3)-VL(CD3) 2 1/2
VH(CD19)-VL(CD19)-VH(CD3)-VL(CD3) 3 3/4
VL(CD19)-VH(CD19)-VL(CD3)-VH(CD3) 4 5/6
VH(CD19)-VL(CD19)-VL(CD3)-VH(CD3) 5 7/8
VL(CD3)-VH(CD3)-VH(CD19)-VL(CD19) 6 9/10
VH(CD3)-VL(CD3)-VH(CD19)-VL(CP19) 7 11/12
VL(CD3)-VH(CD3)-VL(CD19)-VH(CD19) 8 13/14
VH(CD3)-VL(CD3)-VL(CD19)-VH(CD19)
The variable light-chain (VL) and variable heavy-chain (VH) domains
from the HD37 hybridoma (Pezzutto, J. Immunol. 138 (1997), 2793-9)
were cloned according to standard PCR methods (Orlandi, Proc. Natl.
Acad. Sci. USA 86 (1989), 3833-7). cDNA synthesis was carried out
with oligo dT primers and Taq polymerase. For the amplification of
the anti-CD19 V domains via PCR, the primers 5' L1 (SEQ ID NO: 37)
and 3' K (SEQ ID NO: 38), flanking the VL domain, and 5'H1 (SEQ ID
NO: 39) and 3'G (SEQ ID NO: 40) for the heavy chain were used,
based on primers described by Dubel, J. Immunol. Methods 175
(1994), 89-95. The cDNA of the anti-CD3 scFv fragment was kindly
provided by Traunecker (Traunecker, EMBO J. 10 (1991) 3655-9).
Construct 1 as set out in Table 1 was constructed as follows. To
obtain an anti-CD19 scFv-fragment, the corresponding VL- and
VH-regions cloned into separate plasmid vectors, served as
templates for a VL- and VH-specific PCR using the oligonucleotide
primer pairs 5'VLB5RRV (SEQ ID NO: 41)/3'VLGS15 (SEQ ID NO: 42) and
5'VHGS15 (SEQ ID NO: 43)/3'VHBspEI (SEQ ID NO: 28), respectively.
Overlapping complementary sequences were introduced into the
PCR-products that combined to form the coding sequence of 15-amino
acid (Gly.sub.4Ser.sub.1).sub.3-linker during the subsequent
fusion-PCR. This amplification step was performed with the primer
pair 5'VLB5RRV (SEQ ID NO: 41)/3'VHBspE1 (SEQ ID NO: 28) and the
resulting fusion product (or rather anti-CD19 scFv-fragment) was
cleaved with the restriction enzymes. EcoRV and BspE1 and thus
cloned into the bluescript KS-vector (Statagene), containing the
(EcoR1/Sal1-cloned) coding sequence of the anti-17-1A/anti-CD3
bispecific single-chain antibody (actually the version without the
Flag-tag) (Kufer, Cancer Immunol. Immunother. 45 (1997) 193).
Thereby the anti-17-1A-specificity was replaced by the
anti-CD19-scFV-fragment, preserving the 5-amino acid
Gly.sub.4Ser-linker that connects the C-terminal anti-CD3
scFv-fragment. Subsequently, the DNA-fragment encoding the
anti-CD19/anti-CD3 bispecific single-chain antibody with the domain
arrangement VL.sub.CD19-VH.sub.CD19-VH.sub.CD3-VL.sub.CD3 was
subcloned into the EcoR1/Sal1 sites of the described expression
vector pEF-DHFR (Mack, Proc. Natl. Acad. Sci. USA 92 (1995),
7021-5). The resulting plasmid-DNA was transfected into
DHFR-deficient CHO-cells by electroporation. The selection, gene
amplification and protein production were performed as previously
described (Mack, Proc. Natl. Acad. Sci. USA 92 (1995), 7021-5). The
DNA sequence corresponding to construct 1 as set out above in Table
1 is as represented in SEQ ID NO: 29. The protein translation of
this DNA sequence (without leader but including the stop codon) is
as represented in SEQ ID NO: 30.
The remaining constructs as set out above in Table 1 were
constructed as follows. The DNA sequence corresponding to SEQ ID
NO: 29, the protein translation of which (without leader but
including the stop codon) is represented by SEQ ID NO: 30 was used
as PCR template in designing the various anti-CD3/anti-CD19 single
chain bispecific antibodies set out above in Table 1.
To generate a VH-VL arrangement of CD19 in position A1 and A2 (as
defined in FIGS. 1A and 1B), PCR with the respective primer
combination 5'VHCD19BsrGI (SEQ ID NO: 24) and 3'VHCD19GS15 (SEQ ID
NO: 25) or 5'VLCD19GS15 (SEQ ID NO: 26) and 3'VLCD19BspEI (SEQ ID
NO: 27) was used. During these PCR cycles overlapping complementary
sequences were introduced into the PCR-products forming the coding
sequence of a 15 amino acid linker during the subsequent fusion
PCR. The amplified VL and VH domains were fused in a second PCR
reaction (fusion PCR) in which only the outer primers, namely
5'VHCD19BsrGI (SEQ ID NO: 24) and 3'VLCD19BspEI (SEQ ID NO: 27),
and both amplificants were required.
A similar procedure employing other combinations of primers was
used to construct other domain arrangements. A set of appropriate
primers was designed to perform multiple PCR-based cloning steps,
finally resulting in the various VL-VH domain arrangements. The
primer combinations used are listed in the following table:
TABLE-US-00002 TABLE 2 Overview of PCR-based cloning steps used for
construction of positions A1 and A2 of constructs 2, 3, 4, 5, 6, 7
and 8 as shown in Table 1 Resulting PCR PCR N-terminal step Primers
Used step Used Primers Domain order PCR 5'VHCD19BsrGI 3'VHCD19GS15
Fusion 5'VHCD19BsrGI CD 19 A1 (SEQ ID NO: 24) (SEQ ID NO: 25) PCR
(SEQ ID NO: 24) VH-VL PCR 5'VLCD19GS15 3'VLCD19BspEI A1 + A2
3'VLCD19BspEI A2 (SEQ ID NO: 26) (SEQ ID NO: 27) (SEQ ID NO: 27)
PCR 5'VHL2KBsrGI 3'VHL2KGS15 Fusion 5'VHL2KBsrGI Anti-CD3 A1 (SEQ
ID NO: 20) (SEQ ID NO: 21) PCR (SEQ ID NO: 20) VH-VL PCR 5'
VLL2KGS15 3'VLL2KBspEI A1 + A2 3'VLL2KBspEI A2 (SEQ ID NO: 22) (SEQ
ID NO: 23) (SEQ ID NO: 23) PCR 5'VLL2KBsrGI 3'VLL2KGS15 Fusion
5'VLL2KBsrGI Anti-CD3 A1 (SEQ ID NO: 31) (SEQ ID NO: 32) PCR (SEQ
ID NO: 31) VL-VH PCR 5'VHL2KGS15 3'VHL2KBspEI A1 + A2 3'VHL2KBspEI
A2 (SEQ ID NO: 33) (SEQ ID NO: 34) (SEQ ID NO: 34)
In order to change the VH-VL domain arrangement in the C-terminal
position, namely positions B1 and B2 as defined in FIGS. 1A and 1B,
the following primers comprising the designated restriction enzyme
recognition sites were designed to perform the PCR-based cloning
steps.
TABLE-US-00003 TABLE 3 Overview of PCR-based cloning steps used for
construction of positions B1 and B2 of constructs 2, 3, 4, 5, 6, 7
and 8 as shown in Table 1 Resulting C-terminal PCR step Primers
used domain order PCR 5' VLCD19BspEIGS 3' VHCD19BspEI CD 19 VL-VH
B1 + B2 (SEQ ID NO: 19) (SEQ ID NO: 35) 5' VHCD19BspEIGS
3'VLCD19BspEI CD19 VH-VL (SEQ ID NO: 17) (SEQ ID NO: 18) 5'
VLL2KBspEIGS 3'VHL2KBspEI Anti-CD3 (SEQ ID NO: 15) (SEQ ID NO: 16)
VL-VH
The corresponding PCR product, which was flanked by two BspEI
sites, was cloned into a plasmid designated BS-CTI, which was
digested with BspEI and XmaI restriction enzymes. A polylinker
designated CTI (SEQ ID NO: 36) was inserted before into the
Bluescript KS vector (GenBank accession number X52327) using the
restriction enzyme cleavage sites Xbal and SaII in order to provide
additional cleavage sites as well as the sequence encoding a
G.sub.4S linker, six consecutive histidine residues and a stop
codon. During this cloning step the BspEI site of the VH domain was
fused with the XmaI site of the plasmid thereby destroying both
sites. The correct orientation of the variable domain was verified
by sequencing according to standard protocols.
All molecular biological procedures indicated above were carried
out according to standard protocols described in Sambrook,
Molecular Cloning (A Laboratory Manual, 3rd edition, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001).
DNA encoding the single chain bispecific antibodies in Table 1 (SEQ
ID NOs: 29, 1, 3, 5, 7, 9, 11, 13) were transfected into DHFR
deficient CHO cells for eukaryotic protein expression in DHFR
deficient CHO cells as described in Mack et al. (Mack, Proc Natl
Acad Sci USA 92 (1995), 7021-25). Gene amplification of the
construct was induced by increasing concentrations of methotrexate
(MTX) up to a final concentration of 20 nM MTX. The transfected
cells were then expanded and 1 liter of supernatant produced.
EXAMPLE 2
Expression and Purification of the Single Chain Bispecific
Antibodies Directed Against CD3 and CD19
The protein was expressed in chinese hamster ovary cells (CHO).
Transfection of the expression vector was performed following
calcium phosphate treatment of the cells ("Molecular Cloning",
Sambrook et. al. 1989). The cells were grown in roller bottles with
CHO modified DMEM medium (HiQ.RTM., HiClone) for 7 days before
harvest. The cells were removed by centrifugation and the
supernatant containing the expressed protein was stored at
-20.degree. C.
Akta.RTM. FPLC System (Pharmacia) and Unicorn.TM. Software were
used for chromatography. All chemicals were of research grade and
purchased from Sigma D (Deisenhofen) or Merck (Darmstadt).
Immobilized metal affinity chromatography ("IMAC") was performed
using a Fractogel.RTM. column (Merck) which was loaded with
ZnCl.sub.2 according to the protocol provided by the manufacturer.
The column was equilibrated with buffer A2 (20 mM sodium phosphate
buffer pH 7.5, 0.4 M NaCl) and the cell culture supernatant (500
ml) was applied to the column (10 ml) at a flow rate of 3 ml/min.
The column was washed with buffer A2 to remove unbound sample.
Bound protein was eluted using a 2 step gradient of buffer B2 (20
mM sodium phosphate buffer pH 7.5, 0.4 M NaCl, 0.5 M Imidazol)
according to the following: Step 1: 20% buffer B2 in 6 column
volumes; Step 2: 100% buffer B2 in 6 column volumes. Eluted protein
fractions from step 2 were pooled for further purification.
Gel filtration chromatography was performed on a Sephadex S200
HiPrep column (Pharmacia) equilibrated with PBS (Gibco). Eluted
protein samples (flow rate 1 ml/min) were subjected to standard
SDS-PAGE and Western Blot for detection (see FIGS. 4 and 5). Prior
to purification, the column was calibrated for molecular weight
determination (molecular weight marker kit,. Sigma MW GF-200).
Protein concentrations were determined using protein assay dye
(MicroBCA, Pierce) and IgG (Biorad) as standard protein.
The single chain bispecific antibodies were isolated in a two step
purification process of IMAC (FIG. 2) and gel filtration (FIG. 3).
The main product had a molecular weight of ca. 52 kDa under native
conditions as determined by gel filtration in PBS. This molecular
weight corresponds to the single chain bispecific antibody. All
constructs were purified according to this method. Construct #4
could not be purified from cell culture supernatants due to
extremely low levels of specific protein expressed and secreted
into the supernatant.
Purified bispecific protein was analyzed in SDS PAGE under reducing
conditions performed with pre-cast 4-12% Bis Tris gels
(Invitrogen). Sample preparation and application were performed
according to the protocol provided by the manufacturer. The
molecular weight was determined with MultiMark protein standard
(Invitrogen). The gel was stained with colloidal Coomassie
(Invitrogen protocol). The purity of the isolated protein was
>95% as determined by SDS-PAGE (FIG. 4; protein band at 52
kD).
Western Blot was performed using an Optitran.RTM. BA-S83 membrane
and the Invitrogen Blot Module according to the protocol provided
by the manufacturer. The antibodies used were directed against the
His Tag (Penta His, Qiagen) and Goat-anti-mouse Ig labeled with
alkaline phosphatase (AP) (Sigma), and BCIP/NBT (Sigma) as
substrate. The single chain bispecific antibody could be
specifically detected by Western Blot (FIG. 5). A single band was
detected at 52 kD corresponding to the purified bispecific
molecule.
EXAMPLE 3
Flow Cytometric Binding Analysis of CD19.times.CD3 Specific
Polypetides
In order to test the functionality of the construct with regard to
binding capability to CD19 and CD3, a FACS analysis was performed.
For this purpose CD19 positive Nalm 6 cells (human B cell precursor
leukemia) and CD3 positive Jurkat cells (human T cell leukemia)
were used. 200,000 Nalm 6 cells and 200,000 Jurkat cells were
respectively incubated for 30 min on ice with 50 .mu.l of the pure
cell supernatant of CHO cell cultures each expressing bispecific
antibodies with different arrangements of VH and VL domains of CD19
and CD3 (as described in Example 2). The cells were washed twice in
PBS and binding of the construct was detected as follows. The cells
treated as described above were contacted with an unlabeled murine
Penta His antibody (diluted 1:20 in 50 .mu.l PBS with 2% FCS;
Qiagen; Order No. 34660), which specifically binds to cell-bound
construct via the construct's C-terminal histidine tag. A washing
step followed to remove unbound murine Penta His antibody. Bound
anti His antibodies were detected with an Fc gamma-specific
antibody (Dianova, order no. 115-116-071) conjugated to
phycoerythrin, diluted 1:100 in 50 .mu.l PBS with 2% FCS (thick
grey line in FIGS. 6A-6F). As a negative control (thin black line
in FIGS. 6A-6F) fresh culture medium was used in place of culture
supernatant.
Cells were analyzed by flow cytometry on a FACS-Calibur apparatus
(Becton Dickinson, Heidelberg). FACS staining and measuring of the
fluorescence intensity were performed as described in Current
Protocols in Immunology (Coligan, Kruisbeek, Margulies, Shevach and
Strober, Wiley-Interscience, 2002). The binding ability of several
domain arrangements were clearly detectable as shown for example in
FIGS. 6B, 6D and 6F. In FACS analysis all constructs with different
arrangement of VH and VL domains specific for CD19 and CD3 showed
binding to CD3 compared to the negative control using culture
medium and 1. and 2. detection antibody. Strong binding activity
resulting in a shift in fluorescence intensity >5.times.10.sup.1
was observed for the constructs shown in FIG. 6A (#1), B (#2),
D(#6), E (#7), F (#8). Weaker binding to CD3 was observed for
construct # 3 (FIG. 6C). Strong binding to CD19 was observed for
all constructs.
EXAMPLE 4
Bioactivity of Bispecific Antibodies Specific for CD19 and CD3
Cytotoxic activity of the bispecific antibodies with rearranged VH
and VL domains was determined in a fluorochrome release based
cytotoxicity assay.
CD19 positive NALM6 cells were used as target cells
(1.5.times.10.sup.7) labeled with 10 .mu.M calcein AM (Molecular
Probes, Leiden, Netherland, no. C-1430) for 30 min at 37.degree. C.
in cell culture medium. After two washes in cell culture medium,
cells were counted and mixed with the CD4-positive T cell clone
CB15 cells (kindly provided by Dr. Fickenscher, University of
Erlangen/Nuernberg, Germany). 2.times.10.sup.6 CB15 cells and
2.times.10.sup.5 Nalm6 cells were mixed per ml (E:T ratio of 1:10)
and 50 .mu.l of this suspension was used per well in a 96 well
round bottom plate. Antibodies were diluted in RPMI/10% FCS to the
required concentration and 50 .mu.l of this solution was added to
the cell suspension. A standard reaction was incubated at
37.degree. C./5% CO.sub.2 for 2 hours. After the cytotoxic
reaction, the released dye in the incubation medium can be
quantitated in a fluorescence reader (Tecan, Crailsheim, Germany)
and compared with the fluorescence signal from a control reaction
(without bispecific antibody), and the fluorescence signal obtained
for totally lysed cells (for 10 min in 1% saponin). On the basis of
these readings, the specific cytotoxicity was calculated according
to the following formula: [Fluorescence (Sample)-Fluorescence
(Control)]: [Fluorescence (Total Lysis)-Fluorescence
(Control)].times.100.
Sigmoidal dose response curves typically had R.sup.2 values
>0.97 as determined by Prism Software (GraphPad Software Inc.,
San Diego, USA). EC.sub.50 values calculated by the analysis
program were used for comparison of bioactivity.
As shown in FIG. 7 all constructs revealed cytotoxic activity
against CD19 expressing NALM 6 cells. Strongest bioactivity was
observed for constructs #2, 6, 8 and 1. Strong cytotoxic activity
with EC 50 values<500 pg/ml was detected for constructs #2, 6, 8
and 1. In addition to their high bioactivity, constructs #2 and #6
are also especially amenable to inclusion in pharmaceutical
compositions. Constructs #3 and #7 showed EC 50 values of 52 ng/ml
and 31 ng/ml respectively.
EXAMPLE 5
The 145-2C11 Antibody
The monoclonal antibody 145-2C11 directed against murine CD3 was
analysed in different assays in order to characterize this antibody
as group I or II anti-CD3 antibody. 145-2C11 was used by Brissinck,
1991, J. Immunol. 147-4019 for constructing a bispecific antibody
directed against BCL-1 and murine CD3 and also by de Jonge, 1997,
Cancer Immunol. Immunother. 45-162.
5.1. Binding of 145-2C11 to the Recombinant, Purified Extracellular
Domain of the Murine CD3 Epsilon Chain in ELISA
The anti-murine CD3 epsilon antibody (145-2C11 BD biosciences,
Heidelberg, FRG) was coated (50 .mu.l at 5 .mu.g/ml in PBS) on a
Maxisorp ELISA plate (Nunc GmbH, Wiesbaden, FRG). After overnight
incubation unspecific binding was blocked with 1,5% BSA in PBS for
1 hour. After washing three times with 200 .mu.l PBS, different
dilutions of the recombinant C-terminally His6-tagged CD3 protein
(obtained by a procedure analogous to that described in Example 6
for obtaining recombinant human CD3epsilon) and an irrelevant
antigen (BSA) were incubated for 1 hour in the prepared cavities of
the plate. Binding of recombinant CD3 was detected with horseradish
peroxidase conjugated anti-His antibody (Roche Diagnostics GmbH,
Mannheim, FRG; diluted 1:500 in 1.5% BSA in PBS) binding to a
polyhistidine tag. ABTS (2,2'-Azino-di[3-ethylbenzthiazoline
sulfonate (6)] diammonium salt, Roche Diagnostics GmbH, Mannheim,
FRG) was used as substrate according to the specifications of the
manufacturer. The absorbance values were measured on a SPECTRAFluor
Plus photometer (Tecan Deutschland GmbH, Crailsheim). The
measurement wavelength was 405 nm, the reference wavelength was 620
nm. XFLUOR4 Version: V 4.40 for Windows was used as analysis
software. Specific binding of the recombinant, purified
extracellular domain of the murine CD3 epsilon chain to the
145-2C11 antibody was detected for antibody dilutions of 1:5 and
1:25. (FIG. 8).
5.2. Binding of 145-2C11 to a Human T Cell Line Transfected with
the Murine CD3 Epsilon Chain in FACS
Binding of 145-2C11 antibody to Jurkat cells (obtained from ATCC)
transfected with the murine CD3 epsilon chain surface antigen was
tested using an FACS assay. To this end, 2.5.times.10.sup.5 cells
were incubated with a 1:50 dilution of the PE-conjugated 145-2C11
antibody (BD biosciences, Heidelberg, FRG) in 50 .mu.l PBS with 2%
FCS. As a control another sample of cells was incubated with a 1:50
dilution of a PE-conjugated hamster IgG group1 Kappa isotype
control. (BD biosciences, Heidelberg, FRG) in 50 .mu.l PBS with 2%
FCS. Untransfected cells were also assayed under the described
conditions. The samples were measured on a FACSscan (BD
biosciences, Heidelberg, FRG). Specific binding of the 145-2C11
antibody as compared to the isotype control was clearly detectable
on the transfected but not on the untransfected cells (FIGS. 9 and
10) inducing a shift in fluorescence intensity.
5.3. Binding of 145-2C11 to a Murine T Cell Line in FACS
Binding of 145-2C11 antibody to CTLL2 cells (obtained from ATCC)
was tested using an FACS assay. 2.5.times.10.sup.5 cells were
incubated with a 1:50 dilution of the PE-conjugated 145-2C11
antibody (BD biosciences, Heidelberg, FRG) in 50 .mu.l PBS with 2%
FCS. As a control another aliquot of cells was incubated with a
1:50 dilution of a PE-conjugated hamster IgG group1 Kappa isotype
control (BD biosciences, Heidelberg, FRG) in 50 .mu.l PBS with 2%
FCS. The samples were measured on a FACSscan (BD biosciences,
Heidelberg, FRG). Specific binding of the 145-2C11 antibody as
compared to the isotype control was clearly detectable (FIG.
11).
In summary, these data clearly showed that murine anti CD3 antibody
145-2C11 recognized purified recombinant CD3 epsilon as well as
murine CD3 epsilon expressed in eukaryotic cells. 145-2C11 bound to
Jurkat cells transfected with murine CD3 epsilon as well as to a
murine T cell line expressing the CD3 epsilon chain in its native
murine TCR receptor complex. Both cell lines express CD3 epsilon on
the cell surface in the context of other TCR subunits. These two
criteria--binding to purified recombinant CD3 epsilon as well as
binding to cells expressing CD3 epsilon in the TCR complex--were
described as the essential features of anti CD3 antibodies
belonging to "group I" according to the classification described by
Tunnacliffe et al. (Tunnacliffe, 1989, Int. Immunol., 1, 546-550).
In contrast, "group II" antibodies specifically bind to epitopes
the conformations of which are dependent on the whole T cell
receptor complex. According to these definitions 145-2C11 could
clearly be classified as an anti CD3 antibody belonging to "group
I". This confirms the observations of Leo, Proc. Natl. Acad. Sci
USA (1987), 1374, who found that 145-2C11 could still bind to CD3
epsilon when it was dissociated by detergent treatment from the
other chains of the CD3- and T cell receptor-complex, thus
revealing a "group I" CD3 binding pattern.
EXAMPLE 6
Assignment of CD3-reactive Bispecific Single-chain Antibodies to
Different CD3-binding Patterns
CD3-reactive bispecific single-chain antibodies may be assigned to
different CD3-binding patterns according to the classification of
Tunnacliffe, International Immunology 1 (1989), 546. In order to
assign a CD3-reactive bispecific single-chain antibody to the
"group I" CD3-binding pattern an ELISA may be carried out with
purified recombinant human CD3-epsilon. Recombinant human
CD3-epsilon may be e.g. obtained as C-kappa-fusion construct as
described by Tunnacliffe, Immunol. Lett. 21 (1989) 243 or as
truncated soluble CD3-epsilon available according to the following
procedure:
cDNA was isolated from human peripheral blood mononuclear cells.
Preparation of the cells was performed according to standard
protocols (Current Protocols in Immunology (Coligan, Kruisbeek,
Margulies, Shevach and Strober, John Wiley & Sons, Inc., USA,
2002)). The isolation of total RNA and cDNA synthesis by
random-primed reverse transcription was performed according to
standard protocols (Sambrock, Molecular Cloning; Laboratory Manual,
2nd edition, Cold Spring Harbor laboratory Press, Cold Spring
Harbor, N.Y. (1989)). PCR was used to amplify the coding sequence
of the extracellular domain of the human CD3 epsilon chain. The
primers used in the PCR were designed so as to introduce
restriction sites at the beginning and the end of the cDNA coding
for the extracellular portion of the human CD3 epsilon chain (SEQ
ID NO: 80 and SEQ ID NO:81). The introduced restriction sites,
BsrGI and BspEI, were utilised in the following cloning procedures.
The PCR product was then cloned via BsrGI and BspEI into a plasmid
designated BS-Fss-Lsp derived from the Bluescript KS.sup.+ cloning
vector (Stratagene Europe, Amsterdam-Zuiddoost, the Netherlands)
following standard protocols. (The vector was generated by cloning
a DNA fragment (SEQ ID NO: 82) via EcoRI and SaII into Bluescript
KS.sup.+.) The sequence of different clones was determined by
sequencing according to standard protocols. By cloning into
BS-Fss-Lsp the coding sequence of a murine immunoglobulin heavy
chain leader peptide was fused in-frame to the 5' end of the coding
sequence for the extracellular portion of the human CD3 epsilon
chain. The cDNA was then cloned via EcoRI and BspEI into another
plasmid designated as BSCTI to attach a sequence to the C-terminus,
coding for a polyhistidine tag of six consecutive histidine
residues followed by a stop codon (BSCTI is described in Kufer,
Cancer Immunity 1 (2001), 10). In this step the BspEI site of the
cDNA was fused into an XmaI site of the plasmid thereby destroying
both sites. All cloning steps were designed so as to generate an
intact reading frame for the construct. The plasmid now contained a
sequence coding for a protein comprising a murine immunoglobulin
heavy chain leader peptide, to allow for secreted expression,
followed by the extracellular domain of the human CD3 epsilon chain
followed by a polyhistidine tag of six consecutive histidine
residues, to allow for purification and detection via the
polyhistidine tag (SEQ ID NO: 78 and SEQ ID NO: 79). This sequence
was then cloned into the plasmid pFastBac1.TM. (Invitrogen GmbH,
Karlsruhe, FRG) via EcoRI and SalI.
Expression of the extracellular domain of the human CD3 epsilon
chain in High Five.TM. cells was performed using the
Bac-to-Bac.RTM. Baculovirus Expression System (Invitrogen GmbH,
Karlsruhe, FRG) according to the specifications of the
manufacturer. 10 liters of supernatant in batches of 500 ml were
produced. The construct was then purified out of the culture
supernatant. Purification was performed as a two-step purification.
First the diluted supernatants were loaded on ion exchange columns.
The fractionated eluate was tested in an ELISA assay. To this end,
an anti-human CD3 epsilon antibody (UCHT1 BD biosciences,
Heidelberg, FRG) was coated (50 .mu.l at 5 .mu.g/ml in PBS) on a
Maxisorp ELISA plate (Nunc GmbH, Wiesbaden, FRG) overnight.
Unspecific binding was blocked with 1.5% BSA in PBS for 1 hour. All
prior and subsequent washing steps were performed three times with
200 .mu.l PBS. Afterwards, eluate fractions were incubated for 1
hour in the prepared cavities of the plate. Detection of the
recombinant protein was performed with a horseradish peroxidase
conjugated anti-His antibody (Roche Diagnostics GmbH, Mannheim,
FRG; 50 .mu.l of antibody diluted 1:500 in 1.5% BSA in PBS).
Development of the ELISA was performed with ABTS
(2,2'-Azino-bis(3-Ethylbenz-Thiazolin)-6-Sulfonic acid) Roche
Diagnostics GmbH, Mannheim, FRG) according to the specifications of
the manufacturer. Positive fractions were further purified over a
cobalt-chelate column which preferentially binds histidine-tagged
proteins. Eluate fractions were tested using the described ELISA
assay. Positive fractions were pooled and concentrated.
For assignment of CD3-reactive bispecific single-chain antibodies
to the "group I" CD3-binding pattern, purified recombinant human
CD3-epsilon may be coated (50 .mu.l at 10 .mu.g/ml in PBS) on a
Maxisorp ELISA plate (Nunc GmbH, Wiesbaden, FRG) overnight and
unspecific binding subsequently blocked with 1.5% BSA in PBS for 1
hour. Next the ELISA wells are washed three times with 200 .mu.l
PBS. Then purified CD3-reactive bispecific single-chain antibody
(50 .mu.l at 10 .mu.g/ml in 1.5% BSA in PBS) in a version, that (i)
contains an N-terminal FLAG-tag with the amino acid sequence:
dykddddk (obtainable e.g. as described in Mack, PNAS 92 (1995)
7021) but (ii) avoids a polyhistidine tag can be incubated for 1
hour on immobilized CD3-epsilon. As negative control 50 .mu.l 1.5%
BSA in PBS without bispecific single-chain antibody may be used. As
positive control the "group I" anti-CD3 antibody UCHT1 (BD
biosciences, Heidelberg, FRG; 50 .mu.l of antibody diluted to 5
.mu.g/ml in 1.5% BSA in PBS) may be incubated on immobilized
CD3-epsilon. After another washing step carried out as above,
bispecific single-chain antibody specifically bound to human
CD3-epsilon can be detected with an unconjugated anti-FLAG antibody
(ANTI-FLAG M2 obtained from Sigma-Aldrich Chemie GmbH, Taufkirchen
FRG; 50 .mu.l of antibody diluted to 5 .mu.g/ml in 1.5% BSA in PBS)
followed by a horseradish peroxidase-conjugated, goat anti-mouse
IgG, Fc-gamma fragment specific antibody (obtained from Dianova,
Hamburg, FRG; diluted 1:1000 in 50 .mu.l PBS with 1.5% BSA), which
directly detects the control antibody bound to immobilized
CD3-epsilon. Development of the ELISA was carried out with ABTS
(Roche Diagnostics GmbH, Mannheim, FRG) for 90 minutes in
accordance with the specifications of the manufacturer. In contrast
to the control antibody UCHT-1 none of the bispecific single-chain
antibodies based on the CD3-binding specificity described by
Traunecker, EMBO J. 10 (1991) 3655 showed specific interaction with
purified recombinant human CD3-epsilon, thus excluding assignment
to the "group I" CD3-binding pattern. Differentiation between the
"group II" and the "group III" CD3-binding patterns may be carried
out by flowcytometric binding analysis of CD3-reactive bispecific
single-chain antibodies on human T cells and human
CD3-epsilon-transgenic murine T cells as described e.g. in
Tunnacliffe, International Immunology 1(1989) 546. Flowcytometry
may be carried out as described in Example 3 of the present
invention if the bispecific single-chain antibody to be analyzed
carries a polyhistidine-tag or according to the same protocol,
except that the detection antibody is replaced by a
fluorescence-labeled anti-Flag antibody if the bispecific
single-chain antibody is Flag-tagged.
SEQUENCE LISTINGS
1
83 1 1533 DNA Artificial Sequence Description of Artificial
Sequence Synthetic construct 1 tgtacactcc caggtgcagc tgcagcagtc
tggggctgag ctggtgaggc ctgggtcctc 60 agtgaagatt tcctgcaagg
cttctggcta tgcattcagt agctactgga tgaactgggt 120 gaagcagagg
cctggacagg gtcttgagtg gattggacag atttggcctg gagatggtga 180
tactaactac aatggaaagt tcaagggtaa agccactctg actgcagacg aatcctccag
240 cacagcctac atgcaactca gcagcctagc atctgaggac tctgcggtct
atttctgtgc 300 aagacgggag actacgacgg taggccgtta ttactatgct
atggactact ggggccaagg 360 gaccacggtc accgtctcct ccggtggtgg
tggttctggc ggcggcggct ccggtggtgg 420 tggttctgat atccagctga
cccagtctcc agcttctttg gctgtgtctc tagggcagag 480 ggccaccatc
tcctgcaagg ccagccaaag tgttgattat gatggtgata gttatttgaa 540
ctggtaccaa cagattccag gacagccacc caaactcctc atctatgatg catccaatct
600 agtttctggg atcccaccca ggtttagtgg cagtgggtct gggacagact
tcaccctcaa 660 catccatcct gtggagaagg tggatgctgc aacctatcac
tgtcagcaaa gtactgagga 720 tccgtggacg ttcggtggag ggaccaagct
cgagatcaaa tccggaggtg gtggatccga 780 tatcaaactg cagcagtcag
gggctgaact ggcaagacct ggggcctcag tgaagatgtc 840 ctgcaagact
tctggctaca cctttactag gtacacgatg cactgggtaa aacagaggcc 900
tggacagggt ctggaatgga ttggatacat taatcctagc cgtggttata ctaattacaa
960 tcagaagttc aaggacaagg ccacattgac tacagacaaa tcctccagca
cagcctacat 1020 gcaactgagc agcctgacat ctgaggactc tgcagtctat
tactgtgcaa gatattatga 1080 tgatcattac tgccttgact actggggcca
aggcaccact ctcacagtct cctcagtcga 1140 aggtggaagt ggaggttctg
gtggaagtgg aggttcaggt ggagtcgacg acattcagct 1200 gacccagtct
ccagcaatca tgtctgcatc tccaggggag aaggtcacca tgacctgcag 1260
agccagttca agtgtaagtt acatgaactg gtaccagcag aagtcaggca cctcccccaa
1320 aagatggatt tatgacacat ccaaagtggc ttctggagtc ccttatcgct
tcagtggcag 1380 tgggtctggg acctcatact ctctcacaat cagcagcatg
gaggctgaag atgctgccac 1440 ttattactgc caacagtgga gtagtaaccc
gctcacgttc ggtgctggga ccaagctgga 1500 gctgaaacat catcaccatc
atcattagtc gac 1533 2 505 PRT Artificial Sequence Description of
Artificial Sequence Synthetic construct 2 Gln Val Gln Leu Gln Gln
Ser Gly Ala Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile
Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met
Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45
Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50
55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala
Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val
Tyr Phe Cys 85 90 95 Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr
Tyr Tyr Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr
Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Asp Ile Gln Leu Thr 130 135 140 Gln Ser Pro Ala Ser
Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile 145 150 155 160 Ser Cys
Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Leu 165 170 175
Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr 180
185 190 Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro Arg Phe Ser Gly
Ser 195 200 205 Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val
Glu Lys Val 210 215 220 Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr
Glu Asp Pro Trp Thr 225 230 235 240 Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys Ser Gly Gly Gly Gly Ser 245 250 255 Asp Ile Lys Leu Gln Gln
Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 260 265 270 Ser Val Lys Met
Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr 275 280 285 Thr Met
His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 290 295 300
Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 305
310 315 320 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr
Ala Tyr 325 330 335 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala
Val Tyr Tyr Cys 340 345 350 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu
Asp Tyr Trp Gly Gln Gly 355 360 365 Thr Thr Leu Thr Val Ser Ser Val
Glu Gly Gly Ser Gly Gly Ser Gly 370 375 380 Gly Ser Gly Gly Ser Gly
Gly Val Asp Asp Ile Gln Leu Thr Gln Ser 385 390 395 400 Pro Ala Ile
Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys 405 410 415 Arg
Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser 420 425
430 Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser
435 440 445 Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser
Tyr Ser 450 455 460 Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala
Thr Tyr Tyr Cys 465 470 475 480 Gln Gln Trp Ser Ser Asn Pro Leu Thr
Phe Gly Ala Gly Thr Lys Leu 485 490 495 Glu Leu Lys His His His His
His His 500 505 3 1525 DNA Artificial Sequence Description of
Artificial Sequence Synthetic construct 3 tgtacactcc gatatccagc
tgacccagtc tccagcttct ttggctgtgt ctctagggca 60 gagggccacc
atctcctgca aggccagcca aagtgttgat tatgatggtg atagttattt 120
gaactggtac caacagattc caggacagcc acccaaactc ctcatctatg atgcatccaa
180 tctagtttct gggatcccac ccaggtttag tggcagtggg tctgggacag
acttcaccct 240 caacatccat cctgtggaga aggtggatgc tgcaacctat
cactgtcagc aaagtactga 300 ggatccgtgg acgttcggtg gagggaccaa
gctcgagatc aaaggtggtg gtggttctgg 360 cggcggcggc tccggtggtg
gtggttctca ggtgcagctg cagcagtctg gggctgagct 420 ggtgaggcct
gggtcctcag tgaagatttc ctgcaaggct tctggctatg cattcagtag 480
ctactggatg aactgggtga agcagaggcc tggacagggt cttgagtgga ttggacagat
540 ttggcctgga gatggtgata ctaactacaa tggaaagttc aagggtaaag
ccactctgac 600 tgcagacgaa tcctccagca cagcctacat gcaactcagc
agcctagcat ctgaggactc 660 tgcggtctat ttctgtgcaa gacgggagac
tacgacggta ggccgttatt actatgctat 720 ggactactgg ggccaaggga
ccacggtcac cgtctcctcc ggaggtggtg gatccgacat 780 tcagctgacc
cagtctccag caatcatgtc tgcatctcca ggggagaagg tcaccatgac 840
ctgcagagcc agttcaagtg taagttacat gaactggtac cagcagaagt caggcacctc
900 ccccaaaaga tggatttatg acacatccaa agtggcttct ggagtccctt
atcgcttcag 960 tggcagtggg tctgggacct catactctct cacaatcagc
agcatggagg ctgaagatgc 1020 tgccacttat tactgccaac agtggagtag
taacccgctc acgttcggtg ctgggaccaa 1080 gctggagctg aaaggtggtg
gtggttctgg cggcggcggc tccggtggtg gtggttctga 1140 tatcaaactg
cagcagtcag gggctgaact ggcaagacct ggggcctcag tgaagatgtc 1200
ctgcaagact tctggctaca cctttactag gtacacgatg cactgggtaa aacagaggcc
1260 tggacagggt ctggaatgga ttggatacat taatcctagc cgtggttata
ctaattacaa 1320 tcagaagttc aaggacaagg ccacattgac tacagacaaa
tcctccagca cagcctacat 1380 gcaactgagc agcctgacat ctgaggactc
tgcagtctat tactgtgcaa gatattatga 1440 tgatcattac tgccttgact
actggggcca aggcaccact ctcacagtct cctccgggca 1500 tcatcaccat
catcattgag tcgac 1525 4 502 PRT Artificial Sequence Description of
Artificial Sequence Synthetic construct 4 Asp Ile Gln Leu Thr Gln
Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr
Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp
Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro 35 40 45
Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro 50
55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile
His 65 70 75 80 Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln
Gln Ser Thr 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys Gly 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gln Val 115 120 125 Gln Leu Gln Gln Ser Gly Ala
Glu Leu Val Arg Pro Gly Ser Ser Val 130 135 140 Lys Ile Ser Cys Lys
Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met 145 150 155 160 Asn Trp
Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln 165 170 175
Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly 180
185 190 Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met
Gln 195 200 205 Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe
Cys Ala Arg 210 215 220 Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr
Ala Met Asp Tyr Trp 225 230 235 240 Gly Gln Gly Thr Thr Val Thr Val
Ser Ser Gly Gly Gly Gly Ser Asp 245 250 255 Ile Gln Leu Thr Gln Ser
Pro Ala Ile Met Ser Ala Ser Pro Gly Glu 260 265 270 Lys Val Thr Met
Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn 275 280 285 Trp Tyr
Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp 290 295 300
Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly 305
310 315 320 Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala
Glu Asp 325 330 335 Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn
Pro Leu Thr Phe 340 345 350 Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly
Gly Gly Gly Ser Gly Gly 355 360 365 Gly Gly Ser Gly Gly Gly Gly Ser
Asp Ile Lys Leu Gln Gln Ser Gly 370 375 380 Ala Glu Leu Ala Arg Pro
Gly Ala Ser Val Lys Met Ser Cys Lys Thr 385 390 395 400 Ser Gly Tyr
Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg 405 410 415 Pro
Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly 420 425
430 Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr
435 440 445 Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu
Thr Ser 450 455 460 Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr
Asp Asp His Tyr 465 470 475 480 Cys Leu Asp Tyr Trp Gly Gln Gly Thr
Thr Leu Thr Val Ser Ser Gly 485 490 495 His His His His His His 500
5 1528 DNA Artificial Sequence Description of Artificial Sequence
Synthetic construct 5 tgtacactcc caggtgcagc tgcagcagtc tggggctgag
ctggtgaggc ctgggtcctc 60 agtgaagatt tcctgcaagg cttctggcta
tgcattcagt agctactgga tgaactgggt 120 gaagcagagg cctggacagg
gtcttgagtg gattggacag atttggcctg gagatggtga 180 tactaactac
aatggaaagt tcaagggtaa agccactctg actgcagacg aatcctccag 240
cacagcctac atgcaactca gcagcctagc atctgaggac tctgcggtct atttctgtgc
300 aagacgggag actacgacgg taggccgtta ttactatgct atggactact
ggggccaagg 360 gaccacggtc accgtctcct ccggtggtgg tggttctggc
ggcggcggct ccggtggtgg 420 tggttctgat atccagctga cccagtctcc
agcttctttg gctgtgtctc tagggcagag 480 ggccaccatc tcctgcaagg
ccagccaaag tgttgattat gatggtgata gttatttgaa 540 ctggtaccaa
cagattccag gacagccacc caaactcctc atctatgatg catccaatct 600
agtttctggg atcccaccca ggtttagtgg cagtgggtct gggacagact tcaccctcaa
660 catccatcct gtggagaagg tggatgctgc aacctatcac tgtcagcaaa
gtactgagga 720 tccgtggacg ttcggtggag ggaccaagct cgagatcaaa
tccggaggtg gtggatccga 780 cattcagctg acccagtctc cagcaatcat
gtctgcatct ccaggggaga aggtcaccat 840 gacctgcaga gccagttcaa
gtgtaagtta catgaactgg taccagcaga agtcaggcac 900 ctcccccaaa
agatggattt atgacacatc caaagtggct tctggagtcc cttatcgctt 960
cagtggcagt gggtctggga cctcatactc tctcacaatc agcagcatgg aggctgaaga
1020 tgctgccact tattactgcc aacagtggag tagtaacccg ctcacgttcg
gtgctgggac 1080 caagctggag ctgaaaggtg gtggtggttc tggcggcggc
ggctccggtg gtggtggttc 1140 tgatatcaaa ctgcagcagt caggggctga
actggcaaga cctggggcct cagtgaagat 1200 gtcctgcaag acttctggct
acacctttac taggtacacg atgcactggg taaaacagag 1260 gcctggacag
ggtctggaat ggattggata cattaatcct agccgtggtt atactaatta 1320
caatcagaag ttcaaggaca aggccacatt gactacagac aaatcctcca gcacagccta
1380 catgcaactg agcagcctga catctgagga ctctgcagtc tattactgtg
caagatatta 1440 tgatgatcat tactgccttg actactgggg ccaaggcacc
actctcacag tctcctccgg 1500 gcatcatcac catcatcatt gagtcgac 1528 6
503 PRT Artificial Sequence Description of Artificial Sequence
Synthetic construct 6 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser
Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln
Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Trp Pro
Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Lys
Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met
Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90
95 Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp
100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly
Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp
Ile Gln Leu Thr 130 135 140 Gln Ser Pro Ala Ser Leu Ala Val Ser Leu
Gly Gln Arg Ala Thr Ile 145 150 155 160 Ser Cys Lys Ala Ser Gln Ser
Val Asp Tyr Asp Gly Asp Ser Tyr Leu 165 170 175 Asn Trp Tyr Gln Gln
Ile Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr 180 185 190 Asp Ala Ser
Asn Leu Val Ser Gly Ile Pro Pro Arg Phe Ser Gly Ser 195 200 205 Gly
Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Lys Val 210 215
220 Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr Glu Asp Pro Trp Thr
225 230 235 240 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Ser Gly Gly
Gly Gly Ser 245 250 255 Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met
Ser Ala Ser Pro Gly 260 265 270 Glu Lys Val Thr Met Thr Cys Arg Ala
Ser Ser Ser Val Ser Tyr Met 275 280 285 Asn Trp Tyr Gln Gln Lys Ser
Gly Thr Ser Pro Lys Arg Trp Ile Tyr 290 295 300 Asp Thr Ser Lys Val
Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser 305 310 315 320 Gly Ser
Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 325 330 335
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 340
345 350 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser
Gly 355 360 365 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Lys Leu
Gln Gln Ser 370 375 380 Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val
Lys Met Ser Cys Lys 385 390 395 400 Thr Ser Gly Tyr Thr Phe Thr Arg
Tyr Thr Met His Trp Val Lys Gln 405 410 415 Arg Pro Gly Gln Gly Leu
Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg 420 425 430 Gly Tyr Thr Asn
Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr 435 440 445 Thr Asp
Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr 450 455 460
Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His 465
470 475 480 Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val
Ser Ser 485 490 495 Gly His His His His His His 500 7 1531 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
construct 7 tgtacactcc gacattcagc tgacccagtc tccagcaatc atgtctgcat
ctccagggga 60 gaaggtcacc atgacctgca gagccagttc aagtgtaagt
tacatgaact ggtaccagca 120 gaagtcaggc acctccccca aaagatggat
ttatgacaca tccaaagtgg cttctggagt 180 cccttatcgc ttcagtggca
gtgggtctgg gacctcatac tctctcacaa tcagcagcat 240 ggaggctgaa
gatgctgcca cttattactg ccaacagtgg agtagtaacc cgctcacgtt 300
cggtgctggg accaagctgg agctgaaagg tggtggtggt tctggcggcg gcggctccgg
360 tggtggtggt tctgatatca aactgcagca gtcaggggct gaactggcaa
gacctggggc 420 ctcagtgaag atgtcctgca agacttctgg ctacaccttt
actaggtaca cgatgcactg 480 ggtaaaacag aggcctggac agggtctgga
atggattgga tacattaatc
ctagccgtgg 540 ttatactaat tacaatcaga agttcaagga caaggccaca
ttgactacag acaaatcctc 600 cagcacagcc tacatgcaac tgagcagcct
gacatctgag gactctgcag tctattactg 660 tgcaagatat tatgatgatc
attactgcct tgactactgg ggccaaggca ccactctcac 720 agtctcctca
tccggaggtg gtggatccca ggtgcagctg cagcagtctg gggctgagct 780
ggtgaggcct gggtcctcag tgaagatttc ctgcaaggct tctggctatg cattcagtag
840 ctactggatg aactgggtga agcagaggcc tggacagggt cttgagtgga
ttggacagat 900 ttggcctgga gatggtgata ctaactacaa tggaaagttc
aagggtaaag ccactctgac 960 tgcagacgaa tcctccagca cagcctacat
gcaactcagc agcctagcat ctgaggactc 1020 tgcggtctat ttctgtgcaa
gacgggagac tacgacggta ggccgttatt actatgctat 1080 ggactactgg
ggccaaggga ccacggtcac cgtctcctcc ggtggtggtg gttctggcgg 1140
cggcggctcc ggtggtggtg gttctgatat ccagctgacc cagtctccag cttctttggc
1200 tgtgtctcta gggcagaggg ccaccatctc ctgcaaggcc agccaaagtg
ttgattatga 1260 tggtgatagt tatttgaact ggtaccaaca gattccagga
cagccaccca aactcctcat 1320 ctatgatgca tccaatctag tttctgggat
cccacccagg tttagtggca gtgggtctgg 1380 gacagacttc accctcaaca
tccatcctgt ggagaaggtg gatgctgcaa cctatcactg 1440 tcagcaaagt
actgaggatc cgtggacgtt cggtggaggg accaagctcg agatcaaatc 1500
cgggcatcat caccatcatc attgagtcga c 1531 8 504 PRT Artificial
Sequence Description of Artificial Sequence Synthetic construct 8
Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5
10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr
Met 20 25 30 Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg
Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr
Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr
Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys
Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr
Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly 100 105 110 Gly Gly Gly
Ser Gly Gly Gly Gly Ser Asp Ile Lys Leu Gln Gln Ser 115 120 125 Gly
Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys 130 135
140 Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln
145 150 155 160 Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn
Pro Ser Arg 165 170 175 Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp
Lys Ala Thr Leu Thr 180 185 190 Thr Asp Lys Ser Ser Ser Thr Ala Tyr
Met Gln Leu Ser Ser Leu Thr 195 200 205 Ser Glu Asp Ser Ala Val Tyr
Tyr Cys Ala Arg Tyr Tyr Asp Asp His 210 215 220 Tyr Cys Leu Asp Tyr
Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 225 230 235 240 Ser Gly
Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu 245 250 255
Leu Val Arg Pro Gly Ser Ser Val Lys Ile Ser Cys Lys Ala Ser Gly 260
265 270 Tyr Ala Phe Ser Ser Tyr Trp Met Asn Trp Val Lys Gln Arg Pro
Gly 275 280 285 Gln Gly Leu Glu Trp Ile Gly Gln Ile Trp Pro Gly Asp
Gly Asp Thr 290 295 300 Asn Tyr Asn Gly Lys Phe Lys Gly Lys Ala Thr
Leu Thr Ala Asp Glu 305 310 315 320 Ser Ser Ser Thr Ala Tyr Met Gln
Leu Ser Ser Leu Ala Ser Glu Asp 325 330 335 Ser Ala Val Tyr Phe Cys
Ala Arg Arg Glu Thr Thr Thr Val Gly Arg 340 345 350 Tyr Tyr Tyr Ala
Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val 355 360 365 Ser Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 370 375 380
Ser Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu 385
390 395 400 Gly Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val
Asp Tyr 405 410 415 Asp Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile
Pro Gly Gln Pro 420 425 430 Pro Lys Leu Leu Ile Tyr Asp Ala Ser Asn
Leu Val Ser Gly Ile Pro 435 440 445 Pro Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Asn Ile 450 455 460 His Pro Val Glu Lys Val
Asp Ala Ala Thr Tyr His Cys Gln Gln Ser 465 470 475 480 Thr Glu Asp
Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 485 490 495 Ser
Gly His His His His His His 500 9 1531 DNA Artificial Sequence
Description of Artificial Sequence Synthetic construct 9 tgtacactcc
gatatcaaac tgcagcagtc aggggctgaa ctggcaagac ctggggcctc 60
agtgaagatg tcctgcaaga cttctggcta cacctttact aggtacacga tgcactgggt
120 aaaacagagg cctggacagg gtctggaatg gattggatac attaatccta
gccgtggtta 180 tactaattac aatcagaagt tcaaggacaa ggccacattg
actacagaca aatcctccag 240 cacagcctac atgcaactga gcagcctgac
atctgaggac tctgcagtct attactgtgc 300 aagatattat gatgatcatt
actgccttga ctactggggc caaggcacca ctctcacagt 360 ctcctcaggt
ggtggtggtt ctggcggcgg cggctccggt ggtggtggtt ctgacattca 420
gctgacccag tctccagcaa tcatgtctgc atctccaggg gagaaggtca ccatgacctg
480 cagagccagt tcaagtgtaa gttacatgaa ctggtaccag cagaagtcag
gcacctcccc 540 caaaagatgg atttatgaca catccaaagt ggcttctgga
gtcccttatc gcttcagtgg 600 cagtgggtct gggacctcat actctctcac
aatcagcagc atggaggctg aagatgctgc 660 cacttattac tgccaacagt
ggagtagtaa cccgctcacg ttcggtgctg ggaccaagct 720 ggagctgaaa
tccggaggtg gtggatccca ggtgcagctg cagcagtctg gggctgagct 780
ggtgaggcct gggtcctcag tgaagatttc ctgcaaggct tctggctatg cattcagtag
840 ctactggatg aactgggtga agcagaggcc tggacagggt cttgagtgga
ttggacagat 900 ttggcctgga gatggtgata ctaactacaa tggaaagttc
aagggtaaag ccactctgac 960 tgcagacgaa tcctccagca cagcctacat
gcaactcagc agcctagcat ctgaggactc 1020 tgcggtctat ttctgtgcaa
gacgggagac tacgacggta ggccgttatt actatgctat 1080 ggactactgg
ggccaaggga ccacggtcac cgtctcctcc ggtggtggtg gttctggcgg 1140
cggcggctcc ggtggtggtg gttctgatat ccagctgacc cagtctccag cttctttggc
1200 tgtgtctcta gggcagaggg ccaccatctc ctgcaaggcc agccaaagtg
ttgattatga 1260 tggtgatagt tatttgaact ggtaccaaca gattccagga
cagccaccca aactcctcat 1320 ctatgatgca tccaatctag tttctgggat
cccacccagg tttagtggca gtgggtctgg 1380 gacagacttc accctcaaca
tccatcctgt ggagaaggtg gatgctgcaa cctatcactg 1440 tcagcaaagt
actgaggatc cgtggacgtt cggtggaggg accaagctcg agatcaaatc 1500
cgggcatcat caccatcatc attgagtcga c 1531 10 504 PRT Artificial
Sequence Description of Artificial Sequence Synthetic construct 10
Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5
10 15 Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg
Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn
Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Thr Asp
Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr
Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp
Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser
Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile 130 135
140 Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
145 150 155 160 Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser
Gly Thr Ser 165 170 175 Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val
Ala Ser Gly Val Pro 180 185 190 Tyr Arg Phe Ser Gly Ser Gly Ser Gly
Thr Ser Tyr Ser Leu Thr Ile 195 200 205 Ser Ser Met Glu Ala Glu Asp
Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 210 215 220 Ser Ser Asn Pro Leu
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 225 230 235 240 Ser Gly
Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu 245 250 255
Leu Val Arg Pro Gly Ser Ser Val Lys Ile Ser Cys Lys Ala Ser Gly 260
265 270 Tyr Ala Phe Ser Ser Tyr Trp Met Asn Trp Val Lys Gln Arg Pro
Gly 275 280 285 Gln Gly Leu Glu Trp Ile Gly Gln Ile Trp Pro Gly Asp
Gly Asp Thr 290 295 300 Asn Tyr Asn Gly Lys Phe Lys Gly Lys Ala Thr
Leu Thr Ala Asp Glu 305 310 315 320 Ser Ser Ser Thr Ala Tyr Met Gln
Leu Ser Ser Leu Ala Ser Glu Asp 325 330 335 Ser Ala Val Tyr Phe Cys
Ala Arg Arg Glu Thr Thr Thr Val Gly Arg 340 345 350 Tyr Tyr Tyr Ala
Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val 355 360 365 Ser Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 370 375 380
Ser Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu 385
390 395 400 Gly Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val
Asp Tyr 405 410 415 Asp Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile
Pro Gly Gln Pro 420 425 430 Pro Lys Leu Leu Ile Tyr Asp Ala Ser Asn
Leu Val Ser Gly Ile Pro 435 440 445 Pro Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Asn Ile 450 455 460 His Pro Val Glu Lys Val
Asp Ala Ala Thr Tyr His Cys Gln Gln Ser 465 470 475 480 Thr Glu Asp
Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 485 490 495 Ser
Gly His His His His His His 500 11 1528 DNA Artificial Sequence
Description of Artificial Sequence Synthetic construct 11
tgtacactcc gacattcagc tgacccagtc tccagcaatc atgtctgcat ctccagggga
60 gaaggtcacc atgacctgca gagccagttc aagtgtaagt tacatgaact
ggtaccagca 120 gaagtcaggc acctccccca aaagatggat ttatgacaca
tccaaagtgg cttctggagt 180 cccttatcgc ttcagtggca gtgggtctgg
gacctcatac tctctcacaa tcagcagcat 240 ggaggctgaa gatgctgcca
cttattactg ccaacagtgg agtagtaacc cgctcacgtt 300 cggtgctggg
accaagctgg agctgaaagg tggtggtggt tctggcggcg gcggctccgg 360
tggtggtggt tctgatatca aactgcagca gtcaggggct gaactggcaa gacctggggc
420 ctcagtgaag atgtcctgca agacttctgg ctacaccttt actaggtaca
cgatgcactg 480 ggtaaaacag aggcctggac agggtctgga atggattgga
tacattaatc ctagccgtgg 540 ttatactaat tacaatcaga agttcaagga
caaggccaca ttgactacag acaaatcctc 600 cagcacagcc tacatgcaac
tgagcagcct gacatctgag gactctgcag tctattactg 660 tgcaagatat
tatgatgatc attactgcct tgactactgg ggccaaggca ccactctcac 720
agtctcctca tccggaggtg gtggatccga tatccagctg acccagtctc cagcttcttt
780 ggctgtgtct ctagggcaga gggccaccat ctcctgcaag gccagccaaa
gtgttgatta 840 tgatggtgat agttatttga actggtacca acagattcca
ggacagccac ccaaactcct 900 catctatgat gcatccaatc tagtttctgg
gatcccaccc aggtttagtg gcagtgggtc 960 tgggacagac ttcaccctca
acatccatcc tgtggagaag gtggatgctg caacctatca 1020 ctgtcagcaa
agtactgagg atccgtggac gttcggtgga gggaccaagc tcgagatcaa 1080
aggtggtggt ggttctggcg gcggcggctc cggtggtggt ggttctcagg tgcagctgca
1140 gcagtctggg gctgagctgg tgaggcctgg gtcctcagtg aagatttcct
gcaaggcttc 1200 tggctatgca ttcagtagct actggatgaa ctgggtgaag
cagaggcctg gacagggtct 1260 tgagtggatt ggacagattt ggcctggaga
tggtgatact aactacaatg gaaagttcaa 1320 gggtaaagcc actctgactg
cagacgaatc ctccagcaca gcctacatgc aactcagcag 1380 cctagcatct
gaggactctg cggtctattt ctgtgcaaga cgggagacta cgacggtagg 1440
ccgttattac tatgctatgg actactgggg ccaagggacc acggtcaccg tctcctccgg
1500 gcatcatcac catcatcatt gagtcgac 1528 12 503 PRT Artificial
Sequence Description of Artificial Sequence Synthetic construct 12
Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5
10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr
Met 20 25 30 Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg
Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr
Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr
Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys
Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr
Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly 100 105 110 Gly Gly Gly
Ser Gly Gly Gly Gly Ser Asp Ile Lys Leu Gln Gln Ser 115 120 125 Gly
Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys 130 135
140 Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln
145 150 155 160 Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn
Pro Ser Arg 165 170 175 Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp
Lys Ala Thr Leu Thr 180 185 190 Thr Asp Lys Ser Ser Ser Thr Ala Tyr
Met Gln Leu Ser Ser Leu Thr 195 200 205 Ser Glu Asp Ser Ala Val Tyr
Tyr Cys Ala Arg Tyr Tyr Asp Asp His 210 215 220 Tyr Cys Leu Asp Tyr
Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 225 230 235 240 Ser Gly
Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser 245 250 255
Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser 260
265 270 Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Leu Asn Trp Tyr Gln
Gln 275 280 285 Ile Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Asp Ala
Ser Asn Leu 290 295 300 Val Ser Gly Ile Pro Pro Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp 305 310 315 320 Phe Thr Leu Asn Ile His Pro Val
Glu Lys Val Asp Ala Ala Thr Tyr 325 330 335 His Cys Gln Gln Ser Thr
Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr 340 345 350 Lys Leu Glu Ile
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 355 360 365 Gly Gly
Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val 370 375 380
Arg Pro Gly Ser Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala 385
390 395 400 Phe Ser Ser Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Gly
Gln Gly 405 410 415 Leu Glu Trp Ile Gly Gln Ile Trp Pro Gly Asp Gly
Asp Thr Asn Tyr 420 425 430 Asn Gly Lys Phe Lys Gly Lys Ala Thr Leu
Thr Ala Asp Glu Ser Ser 435 440 445 Ser Thr Ala Tyr Met Gln Leu Ser
Ser Leu Ala Ser Glu Asp Ser Ala 450 455 460 Val Tyr Phe Cys Ala Arg
Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr 465 470 475 480 Tyr Ala Met
Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 485 490 495 Gly
His His His His His His 500 13 1528 DNA Artificial Sequence
Description of Artificial Sequence Synthetic construct 13
tgtacactcc gatatcaaac tgcagcagtc aggggctgaa ctggcaagac ctggggcctc
60 agtgaagatg tcctgcaaga cttctggcta cacctttact aggtacacga
tgcactgggt 120 aaaacagagg cctggacagg gtctggaatg gattggatac
attaatccta gccgtggtta 180 tactaattac aatcagaagt tcaaggacaa
ggccacattg actacagaca aatcctccag 240 cacagcctac atgcaactga
gcagcctgac atctgaggac tctgcagtct attactgtgc 300 aagatattat
gatgatcatt actgccttga ctactggggc caaggcacca ctctcacagt 360
ctcctcaggt ggtggtggtt ctggcggcgg cggctccggt ggtggtggtt ctgacattca
420 gctgacccag tctccagcaa tcatgtctgc atctccaggg gagaaggtca
ccatgacctg 480 cagagccagt tcaagtgtaa gttacatgaa ctggtaccag
cagaagtcag gcacctcccc 540 caaaagatgg atttatgaca catccaaagt
ggcttctgga gtcccttatc gcttcagtgg 600 cagtgggtct gggacctcat
actctctcac aatcagcagc atggaggctg aagatgctgc 660 cacttattac
tgccaacagt ggagtagtaa cccgctcacg ttcggtgctg ggaccaagct 720
ggagctgaaa tccggaggtg gtggatccga tatccagctg acccagtctc cagcttcttt
780 ggctgtgtct ctagggcaga gggccaccat ctcctgcaag gccagccaaa
gtgttgatta 840 tgatggtgat agttatttga actggtacca acagattcca
ggacagccac ccaaactcct 900 catctatgat gcatccaatc tagtttctgg
gatcccaccc aggtttagtg gcagtgggtc 960 tgggacagac ttcaccctca
acatccatcc tgtggagaag gtggatgctg caacctatca 1020 ctgtcagcaa
agtactgagg atccgtggac gttcggtgga gggaccaagc tcgagatcaa 1080
aggtggtggt ggttctggcg gcggcggctc cggtggtggt ggttctcagg tgcagctgca
1140 gcagtctggg gctgagctgg tgaggcctgg
gtcctcagtg aagatttcct gcaaggcttc 1200 tggctatgca ttcagtagct
actggatgaa ctgggtgaag cagaggcctg gacagggtct 1260 tgagtggatt
ggacagattt ggcctggaga tggtgatact aactacaatg gaaagttcaa 1320
gggtaaagcc actctgactg cagacgaatc ctccagcaca gcctacatgc aactcagcag
1380 cctagcatct gaggactctg cggtctattt ctgtgcaaga cgggagacta
cgacggtagg 1440 ccgttattac tatgctatgg actactgggg ccaagggacc
acggtcaccg tctcctccgg 1500 gcatcatcac catcatcatt gagtcgac 1528 14
503 PRT Artificial Sequence Description of Artificial Sequence
Synthetic construct 14 Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu
Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Thr Ser
Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Lys Gln
Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro
Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys
Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met
Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly
100 105 110 Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln
Ser Pro Ala Ile 130 135 140 Met Ser Ala Ser Pro Gly Glu Lys Val Thr
Met Thr Cys Arg Ala Ser 145 150 155 160 Ser Ser Val Ser Tyr Met Asn
Trp Tyr Gln Gln Lys Ser Gly Thr Ser 165 170 175 Pro Lys Arg Trp Ile
Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro 180 185 190 Tyr Arg Phe
Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 195 200 205 Ser
Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 210 215
220 Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
225 230 235 240 Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser
Pro Ala Ser 245 250 255 Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile
Ser Cys Lys Ala Ser 260 265 270 Gln Ser Val Asp Tyr Asp Gly Asp Ser
Tyr Leu Asn Trp Tyr Gln Gln 275 280 285 Ile Pro Gly Gln Pro Pro Lys
Leu Leu Ile Tyr Asp Ala Ser Asn Leu 290 295 300 Val Ser Gly Ile Pro
Pro Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 305 310 315 320 Phe Thr
Leu Asn Ile His Pro Val Glu Lys Val Asp Ala Ala Thr Tyr 325 330 335
His Cys Gln Gln Ser Thr Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr 340
345 350 Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly 355 360 365 Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala
Glu Leu Val 370 375 380 Arg Pro Gly Ser Ser Val Lys Ile Ser Cys Lys
Ala Ser Gly Tyr Ala 385 390 395 400 Phe Ser Ser Tyr Trp Met Asn Trp
Val Lys Gln Arg Pro Gly Gln Gly 405 410 415 Leu Glu Trp Ile Gly Gln
Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr 420 425 430 Asn Gly Lys Phe
Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser 435 440 445 Ser Thr
Ala Tyr Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala 450 455 460
Val Tyr Phe Cys Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr 465
470 475 480 Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val
Ser Ser 485 490 495 Gly His His His His His His 500 15 38 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 15 cttccggagg tggtggatcc gacattcagc tgacccag 38 16
25 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 16 cctccggagg agactgtgag agtgg 25 17 38
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 17 cttccggagg tggtggatcc caggtgcagc
tgcagcag 38 18 25 DNA Artificial Sequence Description of Artificial
Sequence Synthetic oligonucleotide 18 cctccggatt tgatctcgag cttgg
25 19 35 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 19 cttccggagg tggtggatcc gatatccagc tgacc
35 20 31 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 20 aggtgtacac tccgatatca aactgcagca g 31
21 50 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 21 ggagccgccg ccgccagaac caccaccacc
tgaggagact gtgagagtgg 50 22 53 DNA Artificial Sequence Description
of Artificial Sequence Synthetic oligonucleotide 22 ggcggcggcg
gctccggtgg tggtggttct gacattcagc tgacccagtc tcc 53 23 25 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 23 aatccggatt tcagctccag cttgg 25 24 31 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 24 aggtgtacac tcccaggtgc agctgcagca g 31 25 50 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 25 ggagccgccg ccgccagaac caccaccacc ggaggagacg
gtgaccgtgg 50 26 53 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 26 ggcggcggcg
gctccggtgg tggtggttct gatatccagc tgacccagtc tcc 53 27 25 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 27 aatccggatt tgatctcgag cttgg 25 28 39 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 28 aatccggagg agacggtgac cgtggtccct tggccccag 39 29
1587 DNA Artificial Sequence Description of Artificial Sequence
Synthetic construct 29 gaattccacc atgggatgga gctgtatcat cctcttcttg
gtagcaacag ctacaggtgt 60 acactccgat atccagctga cccagtctcc
agcttctttg gctgtgtctc tagggcagag 120 ggccaccatc tcctgcaagg
ccagccaaag tgttgattat gatggtgata gttatttgaa 180 ctggtaccaa
cagattccag gacagccacc caaactcctc atctatgatg catccaatct 240
agtttctggg atcccaccca ggtttagtgg cagtgggtct gggacagact tcaccctcaa
300 catccatcct gtggagaagg tggatgctgc aacctatcac tgtcagcaaa
gtactgagga 360 tccgtggacg ttcggtggag ggaccaagct cgagatcaaa
ggtggtggtg gttctggcgg 420 cggcggctcc ggtggtggtg gttctcaggt
gcagctgcag cagtctgggg ctgagctggt 480 gaggcctggg tcctcagtga
agatttcctg caaggcttct ggctatgcat tcagtagcta 540 ctggatgaac
tgggtgaagc agaggcctgg acagggtctt gagtggattg gacagatttg 600
gcctggagat ggtgatacta actacaatgg aaagttcaag ggtaaagcca ctctgactgc
660 agacgaatcc tccagcacag cctacatgca actcagcagc ctagcatctg
aggactctgc 720 ggtctatttc tgtgcaagac gggagactac gacggtaggc
cgttattact atgctatgga 780 ctactggggc caagggacca cggtcaccgt
ctcctccgga ggtggtggat ccgatatcaa 840 actgcagcag tcaggggctg
aactggcaag acctggggcc tcagtgaaga tgtcctgcaa 900 gacttctggc
tacaccttta ctaggtacac gatgcactgg gtaaaacaga ggcctggaca 960
gggtctggaa tggattggat acattaatcc tagccgtggt tatactaatt acaatcagaa
1020 gttcaaggac aaggccacat tgactacaga caaatcctcc agcacagcct
acatgcaact 1080 gagcagcctg acatctgagg actctgcagt ctattactgt
gcaagatatt atgatgatca 1140 ttactgcctt gactactggg gccaaggcac
cactctcaca gtctcctcag tcgaaggtgg 1200 aagtggaggt tctggtggaa
gtggaggttc aggtggagtc gacgacattc agctgaccca 1260 gtctccagca
atcatgtctg catctccagg ggagaaggtc accatgacct gcagagccag 1320
ttcaagtgta agttacatga actggtacca gcagaagtca ggcacctccc ccaaaagatg
1380 gatttatgac acatccaaag tggcttctgg agtcccttat cgcttcagtg
gcagtgggtc 1440 tgggacctca tactctctca caatcagcag catggaggct
gaagatgctg ccacttatta 1500 ctgccaacag tggagtagta acccgctcac
gttcggtgct gggaccaagc tggagctgaa 1560 acatcatcac catcatcatt agtcgac
1587 30 504 PRT Artificial Sequence Description of Artificial
Sequence Synthetic construct 30 Asp Ile Gln Leu Thr Gln Ser Pro Ala
Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys
Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp Ser Tyr Leu
Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu
Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro 50 55 60 Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70
75 80 Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser
Thr 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys Gly 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gln Val 115 120 125 Gln Leu Gln Gln Ser Gly Ala Glu Leu
Val Arg Pro Gly Ser Ser Val 130 135 140 Lys Ile Ser Cys Lys Ala Ser
Gly Tyr Ala Phe Ser Ser Tyr Trp Met 145 150 155 160 Asn Trp Val Lys
Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln 165 170 175 Ile Trp
Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly 180 185 190
Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met Gln 195
200 205 Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala
Arg 210 215 220 Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met
Asp Tyr Trp 225 230 235 240 Gly Gln Gly Thr Thr Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Asp 245 250 255 Ile Lys Leu Gln Gln Ser Gly Ala
Glu Leu Ala Arg Pro Gly Ala Ser 260 265 270 Val Lys Met Ser Cys Lys
Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr 275 280 285 Met His Trp Val
Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 290 295 300 Tyr Ile
Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys 305 310 315
320 Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met
325 330 335 Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr
Cys Ala 340 345 350 Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp
Gly Gln Gly Thr 355 360 365 Thr Leu Thr Val Ser Ser Val Glu Gly Gly
Ser Gly Gly Ser Gly Gly 370 375 380 Ser Gly Gly Ser Gly Gly Val Asp
Asp Ile Gln Leu Thr Gln Ser Pro 385 390 395 400 Ala Ile Met Ser Ala
Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg 405 410 415 Ala Ser Ser
Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly 420 425 430 Thr
Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly 435 440
445 Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu
450 455 460 Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr
Cys Gln 465 470 475 480 Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala
Gly Thr Lys Leu Glu 485 490 495 Leu Lys His His His His His His 500
31 35 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 31 aggtgtacac tccgacattc agctgaccca gtctc
35 32 50 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 32 ggagccgccg ccgccagaac caccaccacc
tttcagctcc agcttggtcc 50 33 53 DNA Artificial Sequence Description
of Artificial Sequence Synthetic oligonucleotide 33 ggcggcggcg
gctccggtgg tggtggttct gatatcaaac tgcagcagtc agg 53 34 30 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 34 aatccggatg aggagactgt gagagtggtg 30 35 25 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 35 cctccggagg agacggtgac cgtgg 25 36 73 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 36 tctagaattc ttcgaatccg gaggtggtgg atccgatatc
cccgggcatc atcaccatca 60 tcattgagtc gac 73 37 36 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 37 gaagcacgcg tagatatckt gmtsacccaa wctcca 36 38 30
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 38 gaagatggat ccagcggccg cagcatcagc 30 39
33 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 39 cagccggcca tggcgcaggt scagctgcag sag
33 40 39 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 40 accaggggcc agtggataga caagcttggg
tgtcgtttt 39 41 37 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 41 aggtgtacac
tccgatatcc agctgaccca gtctcca 37 42 51 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 42
ggagccgccg ccgccagaac caccaccacc tttgatctcg agcttggtcc c 51 43 53
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 43 ggcggcggcg gctccggtgg tggtggttct
caggtsmarc tgcagsagtc wgg 53 44 750 DNA Artificial Sequence
Description of Artificial Sequence Synthetic construct 44
caggtgcagc tgcagcagtc tggggctgag ctggtgaggc ctgggtcctc agtgaagatt
60 tcctgcaagg cttctggcta tgcattcagt agctactgga tgaactgggt
gaagcagagg 120 cctggacagg gtcttgagtg gattggacag atttggcctg
gagatggtga tactaactac 180 aatggaaagt tcaagggtaa agccactctg
actgcagacg aatcctccag cacagcctac 240 atgcaactca gcagcctagc
atctgaggac tctgcggtct atttctgtgc aagacgggag 300 actacgacgg
taggccgtta ttactatgct atggactact ggggccaagg gaccacggtc 360
accgtctcct ccggtggtgg tggttctggc ggcggcggct ccggtggtgg tggttctgat
420 atccagctga cccagtctcc agcttctttg gctgtgtctc tagggcagag
ggccaccatc 480 tcctgcaagg ccagccaaag tgttgattat gatggtgata
gttatttgaa ctggtaccaa 540 cagattccag gacagccacc caaactcctc
atctatgatg catccaatct agtttctggg 600 atcccaccca ggtttagtgg
cagtgggtct gggacagact tcaccctcaa catccatcct 660 gtggagaagg
tggatgctgc aacctatcac tgtcagcaaa gtactgagga tccgtggacg 720
ttcggtggag ggaccaagct cgagatcaaa 750 45 250 PRT Artificial Sequence
Description of Artificial Sequence Synthetic construct 45 Gln Val
Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser 1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20
25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp
Ile 35 40 45 Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn
Gly Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser
Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ala Ser Glu
Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Glu Thr Thr Thr
Val Gly Arg Tyr Tyr Tyr Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly
Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr 130 135 140 Gln
Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile 145 150
155 160 Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr
Leu 165 170 175 Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro Lys Leu
Leu Ile Tyr 180 185 190 Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro
Arg Phe Ser Gly Ser 195 200 205 Gly Ser Gly Thr Asp Phe Thr Leu Asn
Ile His Pro Val Glu Lys Val 210 215 220 Asp Ala Ala Thr Tyr His Cys
Gln Gln Ser Thr Glu Asp Pro Trp Thr 225 230 235 240 Phe Gly Gly Gly
Thr Lys Leu Glu Ile Lys 245 250 46 729 DNA Artificial Sequence
Description of Artificial Sequence
Synthetic construct 46 gatatcaaac tgcagcagtc aggggctgaa ctggcaagac
ctggggcctc agtgaagatg 60 tcctgcaaga cttctggcta cacctttact
aggtacacga tgcactgggt aaaacagagg 120 cctggacagg gtctggaatg
gattggatac attaatccta gccgtggtta tactaattac 180 aatcagaagt
tcaaggacaa ggccacattg actacagaca aatcctccag cacagcctac 240
atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc aagatattat
300 gatgatcatt actgccttga ctactggggc caaggcacca ctctcacagt
ctcctcagtc 360 gaaggtggaa gtggaggttc tggtggaagt ggaggttcag
gtggagtcga cgacattcag 420 ctgacccagt ctccagcaat catgtctgca
tctccagggg agaaggtcac catgacctgc 480 agagccagtt caagtgtaag
ttacatgaac tggtaccagc agaagtcagg cacctccccc 540 aaaagatgga
tttatgacac atccaaagtg gcttctggag tcccttatcg cttcagtggc 600
agtgggtctg ggacctcata ctctctcaca atcagcagca tggaggctga agatgctgcc
660 acttattact gccaacagtg gagtagtaac ccgctcacgt tcggtgctgg
gaccaagctg 720 gagctgaaa 729 47 243 PRT Artificial Sequence
Description of Artificial Sequence Synthetic construct 47 Asp Ile
Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15
Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr 20
25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp
Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn
Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser
Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu
Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His
Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val
Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly 115 120 125 Gly Ser Gly
Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser 130 135 140 Pro
Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys 145 150
155 160 Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys
Ser 165 170 175 Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys
Val Ala Ser 180 185 190 Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser
Gly Thr Ser Tyr Ser 195 200 205 Leu Thr Ile Ser Ser Met Glu Ala Glu
Asp Ala Ala Thr Tyr Tyr Cys 210 215 220 Gln Gln Trp Ser Ser Asn Pro
Leu Thr Phe Gly Ala Gly Thr Lys Leu 225 230 235 240 Glu Leu Lys 48
750 DNA Artificial Sequence Description of Artificial Sequence
Synthetic construct 48 gatatccagc tgacccagtc tccagcttct ttggctgtgt
ctctagggca gagggccacc 60 atctcctgca aggccagcca aagtgttgat
tatgatggtg atagttattt gaactggtac 120 caacagattc caggacagcc
acccaaactc ctcatctatg atgcatccaa tctagtttct 180 gggatcccac
ccaggtttag tggcagtggg tctgggacag acttcaccct caacatccat 240
cctgtggaga aggtggatgc tgcaacctat cactgtcagc aaagtactga ggatccgtgg
300 acgttcggtg gagggaccaa gctcgagatc aaaggtggtg gtggttctgg
cggcggcggc 360 tccggtggtg gtggttctca ggtgcagctg cagcagtctg
gggctgagct ggtgaggcct 420 gggtcctcag tgaagatttc ctgcaaggct
tctggctatg cattcagtag ctactggatg 480 aactgggtga agcagaggcc
tggacagggt cttgagtgga ttggacagat ttggcctgga 540 gatggtgata
ctaactacaa tggaaagttc aagggtaaag ccactctgac tgcagacgaa 600
tcctccagca cagcctacat gcaactcagc agcctagcat ctgaggactc tgcggtctat
660 ttctgtgcaa gacgggagac tacgacggta ggccgttatt actatgctat
ggactactgg 720 ggccaaggga ccacggtcac cgtctcctcc 750 49 250 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
construct 49 Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala Val
Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln
Ser Val Asp Tyr Asp 20 25 30 Gly Asp Ser Tyr Leu Asn Trp Tyr Gln
Gln Ile Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Asp Ala
Ser Asn Leu Val Ser Gly Ile Pro Pro 50 55 60 Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu
Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr 85 90 95 Glu
Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly 100 105
110 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val
115 120 125 Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser
Ser Val 130 135 140 Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser
Ser Tyr Trp Met 145 150 155 160 Asn Trp Val Lys Gln Arg Pro Gly Gln
Gly Leu Glu Trp Ile Gly Gln 165 170 175 Ile Trp Pro Gly Asp Gly Asp
Thr Asn Tyr Asn Gly Lys Phe Lys Gly 180 185 190 Lys Ala Thr Leu Thr
Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met Gln 195 200 205 Leu Ser Ser
Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg 210 215 220 Arg
Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp 225 230
235 240 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 245 250 50 720 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
construct 50 gacattcagc tgacccagtc tccagcaatc atgtctgcat ctccagggga
gaaggtcacc 60 atgacctgca gagccagttc aagtgtaagt tacatgaact
ggtaccagca gaagtcaggc 120 acctccccca aaagatggat ttatgacaca
tccaaagtgg cttctggagt cccttatcgc 180 ttcagtggca gtgggtctgg
gacctcatac tctctcacaa tcagcagcat ggaggctgaa 240 gatgctgcca
cttattactg ccaacagtgg agtagtaacc cgctcacgtt cggtgctggg 300
accaagctgg agctgaaagg tggtggtggt tctggcggcg gcggctccgg tggtggtggt
360 tctgatatca aactgcagca gtcaggggct gaactggcaa gacctggggc
ctcagtgaag 420 atgtcctgca agacttctgg ctacaccttt actaggtaca
cgatgcactg ggtaaaacag 480 aggcctggac agggtctgga atggattgga
tacattaatc ctagccgtgg ttatactaat 540 tacaatcaga agttcaagga
caaggccaca ttgactacag acaaatcctc cagcacagcc 600 tacatgcaac
tgagcagcct gacatctgag gactctgcag tctattactg tgcaagatat 660
tatgatgatc attactgcct tgactactgg ggccaaggca ccactctcac agtctcctca
720 51 240 PRT Artificial Sequence Description of Artificial
Sequence Synthetic construct 51 Asp Ile Gln Leu Thr Gln Ser Pro Ala
Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys
Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Asn Trp Tyr Gln Gln
Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser
Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser 50 55 60 Gly
Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70
75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu
Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly
Gly Ser Gly 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile
Lys Leu Gln Gln Ser 115 120 125 Gly Ala Glu Leu Ala Arg Pro Gly Ala
Ser Val Lys Met Ser Cys Lys 130 135 140 Thr Ser Gly Tyr Thr Phe Thr
Arg Tyr Thr Met His Trp Val Lys Gln 145 150 155 160 Arg Pro Gly Gln
Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg 165 170 175 Gly Tyr
Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr 180 185 190
Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr 195
200 205 Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp
His 210 215 220 Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr
Val Ser Ser 225 230 235 240 52 10 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 52 Gly Tyr Thr
Phe Thr Arg Tyr Thr Met His 1 5 10 53 16 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 53 Tyr Ile Asn
Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Val Lys 1 5 10 15 54 10
PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 54 Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr 1 5 10
55 10 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 55 Arg Ala Ser Ser Ser Val Ser Tyr Met Asn 1 5 10
56 7 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 56 Asp Thr Ser Lys Val Ala Ser 1 5 57 9 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
peptide 57 Gln Gln Trp Ser Ser Asn Pro Leu Thr 1 5 58 10 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
peptide 58 Gly Tyr Ala Phe Ser Ser Tyr Trp Met Asn 1 5 10 59 17 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
peptide 59 Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys
Phe Lys 1 5 10 15 Gly 60 15 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 60 Arg Glu Thr Thr Thr Val
Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr 1 5 10 15 61 15 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 61
Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Leu Asn 1 5 10
15 62 7 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 62 Asp Ala Ser Asn Leu Val Ser 1 5 63 9 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
peptide 63 Gln Gln Ser Thr Glu Asp Pro Trp Thr 1 5 64 372 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
construct 64 caggtgcagc tgcagcagtc tggggctgag ctggtgaggc ctgggtcctc
agtgaagatt 60 tcctgcaagg cttctggcta tgcattcagt agctactgga
tgaactgggt gaagcagagg 120 cctggacagg gtcttgagtg gattggacag
atttggcctg gagatggtga tactaactac 180 aatggaaagt tcaagggtaa
agccactctg actgcagacg aatcctccag cacagcctac 240 atgcaactca
gcagcctagc atctgaggac tctgcggtct atttctgtgc aagacgggag 300
actacgacgg taggccgtta ttactatgct atggactact ggggccaagg gaccacggtc
360 accgtctcct cc 372 65 124 PRT Artificial Sequence Description of
Artificial Sequence Synthetic construct 65 Gln Val Gln Leu Gln Gln
Ser Gly Ala Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile
Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met
Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45
Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50
55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala
Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val
Tyr Phe Cys 85 90 95 Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr
Tyr Tyr Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr
Val Ser Ser 115 120 66 333 DNA Artificial Sequence Description of
Artificial Sequence Synthetic construct 66 gatatccagc tgacccagtc
tccagcttct ttggctgtgt ctctagggca gagggccacc 60 atctcctgca
aggccagcca aagtgttgat tatgatggtg atagttattt gaactggtac 120
caacagattc caggacagcc acccaaactc ctcatctatg atgcatccaa tctagtttct
180 gggatcccac ccaggtttag tggcagtggg tctgggacag acttcaccct
caacatccat 240 cctgtggaga aggtggatgc tgcaacctat cactgtcagc
aaagtactga ggatccgtgg 300 acgttcggtg gagggaccaa gctcgagatc aaa 333
67 111 PRT Artificial Sequence Description of Artificial Sequence
Synthetic construct 67 Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu
Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys Ala
Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp Ser Tyr Leu Asn Trp
Tyr Gln Gln Ile Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr
Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro 50 55 60 Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro
Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr 85 90
95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
105 110 68 45 DNA Artificial Sequence Description of Artificial
Sequence Synthetic oligonucleotide 68 ggtggtggtg gttctggcgg
cggcggctcc ggtggtggtg gttct 45 69 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 69 Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 70 15 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 70 ggaggtggtg gatcc 15 71 5 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 71 Gly Gly Gly
Gly Ser 1 5 72 10 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 72 Ser Ala Ser Ser Ser Val Ser Tyr Met
Asn 1 5 10 73 7 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 73 Asp Thr Ser Lys Leu Ala Ser 1 5 74 9
PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 74 Gln Gln Trp Ser Ser Asn Pro Phe Thr 1 5 75 10
PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 75 Gly Tyr Lys Phe Ser Ser Ser Val Met His 1 5 10
76 17 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 76 Tyr Ile Asn Pro Tyr Asn Asp Val Thr Lys Tyr
Thr Glu Lys Phe Lys 1 5 10 15 Gly 77 11 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 77 Ser Pro Tyr
Tyr Asp Tyr Asp Gly Phe Ala Tyr 1 5 10 78 393 DNA Artificial
Sequence Description of Artificial Sequence Synthetic construct 78
atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt acactccgat
60 ggtaatgaag aaatgggtgg tattacacag acaccatata aagtctccat
ctctggaacc 120 acagtaatat tgacatgccc tcagtatcct ggatctgaaa
tactatggca acacaatgat 180 aaaaacatag gcggtgatga ggatgataaa
aacataggca gtgatgagga tcacctgtca 240 ctgaaggaat tttcagaatt
ggagcaaagt ggttattatg tctgctaccc cagaggaagc 300 aaaccagaag
atgcgaactt ttatctctac ctgagggcaa gagtgtgtga gaactgcatg 360
gagatggatt ccgggcatca tcaccatcat cat 393 79 131 PRT Artificial
Sequence Description of Artificial Sequence Synthetic construct 79
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5
10 15 Val His Ser Asp Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr
Pro 20 25 30 Tyr Lys Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr
Cys Pro Gln 35 40 45 Tyr Pro Gly Ser Glu Ile Leu Trp Gln His Asn
Asp Lys Asn Ile Gly 50 55 60 Gly Asp Glu Asp Asp Lys Asn Ile Gly
Ser Asp Glu Asp His Leu Ser 65 70 75 80 Leu Lys Glu Phe Ser Glu Leu
Glu Gln Ser Gly Tyr Tyr Val Cys Tyr 85 90 95 Pro Arg Gly Ser Lys
Pro Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg 100 105 110 Ala Arg Val
Cys Glu Asn Cys Met Glu Met Asp Ser Gly His His His 115 120 125 His
His His 130 80 36 DNA Artificial Sequence Description of Artificial
Sequence Synthetic primer 80 aggtgtacac tccgatggta atgaagaaat
gggtgg 36 81 42 DNA Artificial Sequence Description of Artificial
Sequence Synthetic primer 81 cgatccggaa tccatctcca tgcagttctc
acacactctt gc 42 82 146 DNA Artificial Sequence Description of
Artificial Sequence Synthetic construct 82 gaattcacca tgggatggag
ctgtatcatc ctcttcttgg tagcaacagc tacaggtgta 60 cactccgata
tcaagcttcc ggacgctccc gactcaagcg cccgtgccac acagccgcaa 120
gatctggcgc cgtgtggtca gtcgac 146 83 10 PRT Artificial Sequence
Description of
Artificial Sequence Synthetic peptide 83 Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser 1 5 10
* * * * *
References